Inhibitors of cyclic amp phosphodiesterases

ABSTRACT

Recombinant fission yeast cells and methods of using them are described, which provide for identification of chemical and biological inhibitors or activators of a target exogenous phosphodiesterase (PDE). The invention provides, in some aspects, compounds that inhibit cAMP PDE activity and compositions that include such compounds. The invention, in part, also includes methods of using cAMP PDE-inhibiting compounds in the treatment of cAMP PDE-associated diseases and/or disorders.

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application 61/124,657, filed Apr. 18, 2008. The entire teachings of the referenced provisional application are incorporated by reference herein in its entirety.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by grant No. 1 R21 GM079662-01 from the National Institute of General Medical Sciences of the National Institute of Health (NIH). The Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention provides methods for treating inflammatory diseases comprising either the administration of dual phosphodiesterase 4-phosphodiesterase 7 (PDE4/PDE7) inhibitors, or the simultaneous or sequential co-administration of selective PDE4 inhibitors together with selective PDE7 inhibitors. The present invention further relates to pharmaceutical compositions containing these inhibitors, and the use of these inhibitors in the treatment of inflammatory diseases.

BACKGROUND OF THE INVENTION

Phosphodiesterases (PDEs) play an important role in various biological processes by hydrolysing the key second messengers adenosine and guanosine 3′,5′-cyclic monophosphates (cAMP and cGMP respectively) into their corresponding 5′-monophosphate nucleotides. Therefore, inhibition of PDE activity produces an increase of cAMP and cGMP intracellular levels that activate specific protein phosphorylation pathways involved in a variety of functional responses. At least 11 families of PDEs exist, some of which (PDE 4, 7, 8) are specific for cAMP, and others (PDE 5, 6, 9) for cGMP, while other family members have dual specificity (PDE 1, 2, 3, 10, 11). PDEs are expressed in a tissue and cell specific manner, and expression also changes depending on the cell state. For example, resting T lymphocytes express mainly PDE3 and PDE4. However, upon activation, T cells dramatically upregulate PDE7 and appear to rely on this isozyme for regulation of cAMP levels.

Three PDE1 genes have been identified and are expressed in heart, lung, and kidney tissue, as well as in circulating blood cells and smooth muscle cells. PDE2 is expressed in adrenal gland, heart, lung, liver, and platelets. The PDE3 family, expressed by the PDE3A and PDE3B genes, are distributed in several tissues including the heart, lung, liver, platelets, adipose tissue, and inflammatory cells. Over twenty isoforms of PDE4 are expressed by four genes, and these are expressed in a wide variety of tissues including heart, kidney, brain, liver, lung, the gastrointestinal track and circulating blood and inflammatory cells. PDE5 (three isoforms) is expressed for example in the human corpus cavernosum (vascular) smooth muscle, lung, and platelets. PDE6 is expressed by three genes in photoreceptors of the retina. PDE7 proteins are expressed by two genes in skeletal muscle, heart, kidney, brain, pancreas, and T lymphocytes. PDE8 is expressed by two genes in testes, eye, liver, skeletal muscle, heart, kidney, ovary, brain, and T lymphocytes. PDE9 is expressed in kidney, liver, lung, and brain. PDE10 is expressed in the testes as well as the brain. PDE11 is expressed in skeletal muscle, prostate, kidney, liver, pituitary and salivary glands, and testes (Boswell-Smith V. et al., 2006, Brit J Pharm 147:S252-57).

The four PDE4 subfamilies are encoded by separate genes (A, B, C, D) that generate many isoforms through the use of alternative mRNA splicing and distinct promoters. Isoforms generated by the four PDE4 subfamilies are each individually characterized by unique N-terminal regions. They can be divided into long forms, which possess both the Upstream Conserved Region 1 (UCR1) and Upstream Conserved Region (UCR2) regulatory regions, while the short isoforms lack UCR1 and the super-short isoforms lack UCR1 and also have a truncated UCR2.

Two PDE7 genes (PDE7A and PDE7B) have been identified. PDE7A has three isoforms generated by alternate splicing; PDE7A1 is restricted mainly to T cells and the brain, PDE7A2 for which mRNA is expressed in a number of cell types including muscle cells, and PDE7A3 found in activated T cells. The PDE7A1 and PDE7A2 isoforms have different sequences at the amino termini. PDE7A3 is similar to PDE7A1 in the amino terminus but has a different carboxy terminal sequence than PDE7A1 and PDE7A2. PDE7B has approximately 70% homology to PDE7A in the enzymatic core.

PDEs are important drug targets. Many PDE-specific inhibitors have been developed and are currently being used or are being evaluated for use, such as KS-505a (PDE1); EHNA (PDE2); Cilostamide, Enoxamone, Milrinone, Siguazodan (PDE3); Rolipram, Roflumilast, Cilomilast (PDE4); Sildenafil, Zaprinast (PDE5); Dipyridamole (PDE6); BRL-50481 (PDE7), BAY 73-6691 (PDE9) (Boswell-Smith V. et al., 2006, Brit J Pharm 147:S252-57).

PDE2 inhibitors were developed for the treatment of sepsis, and Acute Respiratory Distress Syndrome (ARDS).

PDE3 inhibitors were developed for the treatment of congestive heart failure, airway diseases, and to treat fertility. PDE3 inhibitors have been shown to relax vascular and airway smooth muscle, inhibit platelet aggregation and induce lipolysis.

PDE4 inhibitors were developed for the treatment of inflammatory airways disease, asthma, chronic obstructive pulmonary disease (COPD), allergic rhinitis, psoriasis, rheumatoid arthritis, depression, schizophrenia, Alzheimer's Disease, memory loss, cancer, dermatitis and multiple sclerosis. Inhibition of PDE4 has been associated with an anti-inflammatory response associated with T cells as well as monocytes, macrophages, mast cells, basophils and neutrophils. The majority of PDE4 selective inhibitors reported on to date serve to inhibit PDE4 isoforms from all four subfamilies with either little or no PDE4 subfamily selectivity, while PDE4A and PDE4B are the actual anti-inflammatory targets.

PDE5 inhibitors were developed for the treatment of erectile dysfunction and impotence, pulmonary hypertension, female sexual dysfunction, cardiovascular disease, premature ejaculation, stroke, leukaemia, and renal failure.

PDE7 inhibitors were developed for the treatment of inflammation. Increasing cAMP levels by selective PDE7 inhibition appears to be a potentially promising approach to specifically block T-cell mediated immune responses.

There are side-effects associated with many PDE inhibitors, which limit their use. PDE1 inhibitors have demonstrated potent vasodilator activity. PDE3 inhibitors have demonstrated potent cardiac inotropic activity. Nausea, emesis and cardiac arrhythmias remain the major obstacles in the development of PDE4 inhibitors, especially caused by inhibition of PDE4D. PDE5 inhibitors affect PDE6 activity in the photoreceptors of the retina and can lead to visual disturbances consisting of altered color perception. There is an unmet medical need to develop effective methods and identify effective PDE inhibitor compounds, including PDE inhibitors that specifically act on individual family members and even on individual isoforms expressed from a single PDE gene, for treatment of immune and inflammatory disorders.

SUMMARY OF THE INVENTION

Described herein are PDE4 inhibitors (e.g., PDE4A inhibitors, PDE4B inhibitors), PDE7 inhibitors, combination inhibitors (e.g., PDE4A/4B, PDE4/7, such as PDE4A/7, PDE4B/7, PDE4A/4B/7); methods in which such inhibitors are used, including methods in which an inhibitor is used to treat a condition or disease (e.g., an inflammatory disease, a neurological disease, memory loss, chronic lymphocytic leukemia, osteoporosis, HIV infection, cerebrovascular ischemia); and pharmaceutical compositions comprising at least one PDE4 inhibitor (e.g., PDE4A inhibitor, PDE4B inhibitor), PDE7 inhibitor, PDE4/7 combination inhibitor) and an appropriate carrier. The pharmaceutical composition can optionally additionally comprise at least one additional drug.

PDE inhibitors were identified using methods described herein, such as high throughput drug screens on genetically engineered fission yeast strains that express drug targets (e.g., PDE4A and/or PDE4B, which are anti-inflammatory targets). PDE inhibitors were identified based on their ability to stimulate growth and compounds were identified because they were effective in live cells. In addition, targets used in the assays are full-length proteins (as opposed to simply the catalytic domain) and the assay used included a built-in toxicity test, permeability test and stability test. The inhibitors identified display a very high degree of target specificity. Compounds identified include inhibitors that act on two of four PDE4 family enzymes and inhibitors that act on combinations of PDE4 and PDE7 strains. One example of a compound identified is compound BC58, which is an effective PDE4A/4B inhibitor that exhibits limited/essentially no inhibition of PDE4D. Limited inhibition of PDE4D by a PDE inhibitor is desirable, in view of the fact that inhibition of PDE4D causes emesis and cardiac arrhythmias. Subtype specificity was confirmed by means of cAMP assays.

As described herein and as shown in the tables, Applicant has identified compounds that are PDE4A inhibitors; PDE4B inhibitors; PDE4A/4B inhibitors; PDE7 inhibitors; and combination PDE4/7 inhibitors (PDE4A/7, PDE4B/7, PDE4A/4B/7). Inhibitors described herein can be used individually (e.g., a PDE4A inhibitor; a PDE4B inhibitor; a PDE7 inhibitor; a combination inhibitor, such as PDE4A/7, PDE4B/7, PDE4A/4B/7 or in combination with one or more other PDE inhibitor(s) (e.g., PDE4A inhibitor with a PDE4B inhibitor and/or a PDE7 inhibitor) or in combination with another therapeutic agent/drug that is also a PDE inhibitor or another therapeutic agent/drug that is not a PDE inhibitor.

Co-administration of PDE inhibitors, which may be selective for the PDE family, a specific PDE subfamily, or a specific isoform of a PDE-subfamily member, such as a selective PDE4 inhibitor with a selective PDE7 inhibitor, or administration of a dual PDE4/7 inhibitor (PDE4A/7, PDE4B/7, PDE4A/4B/7), can be used to increase therapeutic effectiveness, and/or reduce toxicity and/or side effects (such as nausea) over presently-available approaches. The combined activity of PDE4 and PDE7 or dual PDE4/7 inhibitors may be especially useful in treating a wide variety of immune and inflammatory disorders as an immunosuppressant therapy. PDE7 inhibitors act by inhibiting a very early stage of the T cell activation cascade. PDE4 inhibition decreases the production of the pro-inflammatory cytokines such as Tumor Necrosis Factor alpha (TNF-α) in monocytes and macrophages, as well as affect granulocytes, such as neutrophils. Dual PDE4/7 inhibitors or co-administration of selective PDE4 and PDE7 inhibitors are expected to be particularly useful in treating disorders that involve one or more inflammatory response alleviated, at lease in part, by PDE4 inhibition (e.g., via decreased mast cell, basophil and neutrophil degranulation and monocyte and macrophage production of pro-inflammatory cytokines such as TNF-α), and/or are alleviated at least in part by PDE7 inhibition (e.g., through decreased T cell activation), e.g., disorders such as rheumatoid arthritis, inflammatory bowel disease (IBD), psoriasis, asthma, chronic obstructive pulmonary disease (COPD), lupus, visceral pain, osteoarthritis, osteoporosis, allergic rhinitis, cancer, acquired immune deficiency syndrome, allergy, fertility diseases, and multiple sclerosis among others. A PDE4-PDE7 inhibitor combination is also expected to have a decreased potential for clinically significant side effects compared to current immunosuppressants.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows growth of fission yeast strains carrying mutations in the adenylate cyclase (git2) gene, the PDE (cgs2) gene, or the git1 (a regulator of adenylate cyclase) gene on various growth media. The arrows point to two strains that demonstrate that a reduction in PDE activity can restore 5FOA-resistant growth to either a git2-7 or git1-1 mutant strain. Note that the git2 deletion strain (git2 Δ) remains 5FOA-sensitive even when carrying the cgs2-s1 mutation.

FIG. 2 shows β-galactosidase activity resulting from fbp1-lacZ expression as a function of time after removal of cAMP from the growth medium. β-galactosidase activity was measured at the time points indicated after cells were transferred from EMM medium containing 5 mM cAMP to EMM without cAMP.

FIG. 3 shows schematic diagrams of cAMP-regulated growth phenotypes in fission yeast strains expressing the fbp1-ura4 reporter. FIG. 3A is a diagram showing that glucose signaling leads to adenylylcyclase activation and a cAMP signal, which activates PKA to repress fbp1-ura4 transcription. These cells cannot grow in medium lacking uracil (-Ura), but do grow in medium containing 5FOA. FIG. 3B is a diagram showing that cells carrying mutations in genes required for glucose signaling have reduced adenylylcyclase activity to lower cAMP levels. This results in low PKA activity and a failure to repress fbp1-ura4 transcription. These cells grow in medium lacking uracil (-Ura), but do not grow in medium containing 5FOA. FIG. 3C is a diagram showing a screen for PDE activators carried out by taking a strain such as the one in panel A and screening for compounds that enhance growth in medium lacking uracil. The compounds identified include ones that stimulate PDE activity to lower cAMP levels. FIG. 3D is a diagram showing a screen for PDE inhibitors carried out by taking a strain such as the one in FIG. 3B and screening for compounds that enhance growth in 5FOA medium. The compounds identified include ones that inhibit PDE activity to raise cAMP levels.

FIG. 4 is a graph showing that deletion of pap1⁺ enhances rolipram-mediated fbp1-lacZ repression. β-galactosidase activity from two independent exponential phase cultures was determined in pap1⁺ (light gray bars) and pap1Δ (dark gray bars) gpa2⁻ mutant strains grown in EMM complete medium containing various concentrations of rolipram as indicated, while receiving identical volumes of DMSO (vehicle). Values are plotted as a percent of the vehicle-treated cultures that did not receive rolipram. The ratio of fold-inhibition in the pap1Δ strain versus the pap1⁺ strain is shown for each concentration of rolipram.

FIGS. 5A and 5B show graphs demonstrating that PDE inhibitors alter cAMP levels in yeast strains. FIG. 5A shows results when cAMP levels were measured in exponential phase cells immediately prior to 200 μM drug addition (rolipram for strains CHP1085 (PDE4A) and CHP1114 (PDE4B), and EHNA for strain LWP371 (PDE2A)), and 10, 30, 60, and 120 minutes after drug addition. Values represent the average and SD of two or three independent experiments. FIG. 5B shows results when cAMP levels were measured 60 minutes after addition of either vehicle (DMSO), 20 μM drug, or 200 μM drug as indicated. The strains used are as in FIG. 5A, together with strain CHP1141 (PDE8A). Values represent the average and SD of two or three independent experiments.

FIGS. 6A and 6B show the in vitro assay of BC54 action. Lymphoma cells are treated with the drug for 24 hours, then stained with Hoechst dye. The cells form a clearly defined population on the fsc/ssc dot plot. Vincristine treatment was used as a positive control. Compound BC54 induces apoptosis in cell culture of tumor cells (almost 100% of “live” population is gone). BC54 is referred to in the figure as S54.

FIGS. 7A and 7B show results of an in vivo assay of BC54 action. The results show that BC54 reduces the tumor load in lymph nodes of treated mice who have received a transplanted tumor that grows rapidly over the course of a week. No significant effect of BC54 was shown on spleen tumor burden.

FIGS. 8A, 8B and 8C show the effect of BC54 on the proliferation and viability of PBMC cells. BC54 was not found to be very toxic to the cells.

FIG. 9 shows the assessment of antiviral activity of BC54. PBMC cells were isolated from uninfected rhesus macaques and mitogen stimulated (PHA). The cells were then incubated with SIVmac239 for 2 hours, following which they were washed to remove any free virus. BC54 was then added to the infected cells. Fresh compound was added after 3 days. The antiviral activity was measured by SIV p27 ELISA on day 3 and 7, while the cell viability was determined on day 7 by MTT assay. The treatment of cells on Day 1 and Day 3, leads to a 30-fold and 100-fold reduction in viral load on Days 4 and 7, respectively.

FIG. 10 shows the 5FOA growth response by PDE7A- (left panel) and PDE7B-expressing (right panel) fission yeast strains. PDE inhibition allows growth in 5FOA medium as measured by optical density after 48 hours incubation at 30° C. Compound BC12 and six structural analogs were tested for growth stimulation. For both PDE7A and PDE7B, BC12 is the most potent inhibitor, promoting growth at low micromolar concentrations.

FIG. 11 is the in vitro enzyme assay data to demonstrate that BC12 stimulates PDE7B activity in vitro. The PDE7B catalytic domain was expressed in and purified from E. coli and tested for activity in an in vitro enzyme assay, measuring the hydrolysis of cAMP to AMP. Four dilutions of the enzyme preparation were tested at various compound concentrations (X axis). The percent of cAMP hydrolyzed is plotted on the Y axis. The addition of compound BC12 increases cAMP hydrolysis. There is no evidence that PDE7B is activated in vivo by BC12, however this result suggests that BC12 does not act by occupying the cAMP-binding site of the enzyme.

FIG. 12 shows the effect of BC12, BC28, BC54, and BC58 relative to rolipram (PDE4 inhibitor) and BRL50481 (PDE7 inhibitor) on IL2 secretion by concavalin A treated Jurkat cells.

FIG. 13 is the in vitro enzyme assays of PDE7B activity in extracts made from rat pulmonary endothelial cells. BC12 and BC28 inhibit with KI values that are approximately 100-fold lower than that of the GlaxoSmithKline compound BRL50481. BC12 inhibits PDE7B, unlike the activation seen with the E. coli-expressed enzyme. KI for BC12 and BC28 on PDE7 from rat pulmonary endothelial cells is approximately 200 nM, while the KI on PDE4 is about 25-30 micromolar.

Also included are Tables 1 through 19.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are methods for treating a wide variety of immune and inflammatory disorders using PDE4 inhibitor(s), PDE7 inhibitor(s); a combination of PDE4 inhibitors and PDE7 inhibitors, or dual PDE4A/4B, or dual PDE4/7 inhibitors (e.g., PDE4A/7, PDE4B/7, PDE4A/4B/7 inhibitors), which may be selective for the PDE family, a specific PDE subfamily, or a specific isoform of a PDE-subfamily member. Also described are compounds and compositions that include at least one PDE4 inhibitor (e.g., a PDE4A inhibitor, a PDE4B inhibitor); at least one PDE7 inhibitor, at least one combination inhibitor (e.g., PDE4A/4B inhibitor, PDE4/7 inhibitor, such as PDE4A/7 inhibitor, PDE4B/7 inhibitor, PDE4A/4B/7 inhibitor) or a combination of two or more such inhibitors. Such compositions may also include a pharmaceutically acceptable carrier. When administered to an individual, the compounds inhibit PDE4 and/or PDE7 activity in vivo and are useful for treating immune and inflammatory disorders. The selective PDE4 or PDE7 inhibitor compounds described herein, used alone or in combination, and dual PDE4/7 inhibitors may be used. Combinations (e.g., combinations of two or more PDE4 inhibitors (e.g., PDE4A inhibitor and PDE4B inhibitor); combinations of one or more PDE4 inhibitor with a PDE7 inhibitor) may be more effective than either a selective PDE4 inhibitor or a selective PDE7 inhibitor administered alone in the treatment of disease, through additive or synergistic activity resulting from the combined inhibition of PDE4 and PDE7. Expression of PDE7A, for example, increases when PDE4 is inhibited.

Described herein are compounds that exhibit low toxicity against biological organisms in vitro. In some embodiments the compounds exhibit the ability to permeate biological organisms in vitro, e.g., to cross a biological membrane. In some embodiments the compounds exhibit high bio-stability in biological organisms in vitro, e.g., are not rapidly degraded or are active for an extended period.

There are numerous compounds described herein. They are grouped into Groups NI-NIII, as shown below. In certain embodiments the compounds are selected from compounds of formula (NI) (Group NI).

In one set of embodiments, the compound may have a structure as in formula NI,

wherein:

R⁷ and R⁸ can be the same or different and are optionally substituted carbonyl groups; and

R⁹ is alkyl.

In some embodiments, R⁷ is C═OO)R^(a), wherein R^(a) is optionally substituted alkyl; R⁸ is (C═O)R^(b), wherein R^(b) is optionally substituted alkyl or optionally substituted arylalkyl; and R⁹ is cyclohexyl. For example, the compound may have the structure,

In one embodiment, the compound has the following structure, also referred to herein as “BC54”:

Results described herein show that BC54 is a good PDE4 and PDE7 combination inhibitor, but does not act on all PDEs (no effect on PDE8A or PDE3A). It shows good activity in TNFα and CLL assays. It is effective in mouse DLBCL model and in SIV cell culture. It affects PDE4D, but is weaker than on other 4 s and 7 s.

In another set of embodiments, the compound may have a structure as in formula NII,

wherein:

R¹⁰ is an aryl group, optionally substituted; and

R¹¹, R¹², R¹³, R¹⁴, and R¹⁵ can be the same or different and are hydrogen, halide, or alkyl.

In some embodiments, R¹⁰ is phenyl, optionally substituted with N(CH₃)₂ or NO₂; R¹¹, R¹², R¹⁴, R¹⁵ are hydrogen; and R¹³ is methyl, chloro, fluoro, or bromo. For example, the compound may have the structure,

In one embodiment, the compound has the following structure, also referred to herein as “BC12”:

Results described herein show that BC12 is a PDE7 inhibitor that appears to activate PDE4D and PDE7B in vitro (not in vivo). It is also potent inhibitor of PDE7B from pulmonary endothelial cells. It shows activity in TNFα & CLL assays, and has an apoptosis effect on lymphoma cell line.

In another set of embodiments, the compound may have a structure as in formula NIII,

wherein:

is a single bond or a double bond;

R¹⁶ and R¹⁷ can be the same or different and are hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heterocycle, or an optionally substituted carbonyl group;

or, R¹⁶ and R¹⁷ are joined together to form a ring, optionally substituted;

R¹⁸ is alkyl;

R¹⁹ is hydrogen, optionally substituted alkyl, or an optionally substituted carbonyl group;

or, R¹⁸ and R¹⁹ are joined together to form a ring, optionally substituted;

R²⁰ is absent or hydrogen, provided that, when

is a single bond, R²⁰ is hydrogen, and, when

is a double bond, R²⁰ is absent; and

R²¹ is ═S, optionally substituted alkyl, optionally substituted heteroalkyl, or optionally substituted arylalkyl.

In some embodiments, R¹⁸ is methyl.

In some embodiments, R¹⁶ and R¹⁷ are joined together to form an a cycloalkyl or heterocycle ring, optionally substituted. In some cases, R¹⁶ and R¹⁷ may be joined to form a 5-, 6-, 7-, or 8-membered ring, optionally substituted. For example, R¹⁶ and R¹⁷ may be joined to form a morpholinyl, piperidinyl (e.g., methyl-piperidinyl), piperazinyl, substituted derivatives thereof, and the like. In some embodiments, R¹⁸ and R¹⁹ are joined together to form a cycloalkyl or heterocycle ring, optionally substituted. In some cases, R¹⁸ and R¹⁹ may be joined to form a 5-, 6-, 7-, or 8-membered ring, optionally substituted. For example, R¹⁸ and R¹⁹ may be joined to form a 5- or 6-membered carbon ring fused to the parent structure.

In some embodiments, R¹⁹ is an alkyl group, such as methyl or ethyl. In some embodiments, R¹⁹ is a carbonyl group, such an ester or an amide, optionally substituted. The nitrogen of the amide group may comprise one or more substituents. In some cases, R¹⁹ is a group having the formula, (C═O)NHR^(c), where R^(c) is an optionally substituted alkyl or optionally substituted aryl group. For example, R^(c) may be substituted with one or more halogens (e.g., Cl, Br, F), alkoxy groups (e.g., OCH₃), alkyl groups, aryl groups, heterocycles, carbonyl groups, substituted derivatives thereof (e.g., CF₃), and the like. In some cases, R^(c) may include a polycyclic ring system, such as a phenyl group fused to another ring (e.g., a 1,4-dioxane ring).

In some embodiments, the compound may have the structure,

In one embodiment, the compound has the following structure, also referred to herein as “BC28”:

Results described herein show that BC28 is a potent PDE7B inhibitor (200 nM on PDE7B from pulmonary endothelial cells) and induced cAMP elevation in a yeast PDE7B-expressing strain. BC28 shows TNFα synergy with PDE4 inhibitors, though not as strong as for BC30. BC28 had strong reduction in IL2 expression from Jurkat cells.

In one embodiment, the compound has the following structure, also referred to herein as “BC33”:

Results described herein show that BC33 is a PDE4 inhibitor. It also shows some activity on PDE4D3 (but not 4D2).

In one embodiment, the compound has the following structure, also referred to herein as “BC44”:

Results described herein show that BC44 is very good PDE4 and PDE7 inhibitor, with low PDE4D inhibition.

In one embodiment, the compound has the following structure, also referred to herein as “BC58”:

Results described herein show that BC58 is PDE4A/4B-specific inhibitor. It shows good PDE4 inhibitor activity is TNFα assay.

In one embodiment, the compound has the following structure, also referred to herein as “BC64”:

Results described herein show that BC64 is a moderate PDE7 inhibitor.

As used herein, the terms “alkyl,” “alkenyl” and the prefix “alk-” are inclusive of both straight chain and branched chain groups and of cyclic groups, i.e. cycloalkyl and cycloalkenyl. Unless otherwise specified, these groups contain from 1 to 20 carbon atoms, with alkenyl groups containing from 2 to 20 carbon atoms. Preferred groups have a total of up to 10 carbon atoms. Cyclic groups can be monocyclic or polycyclic and preferably have from 3 to 10 ring carbon atoms. Exemplary cyclic groups include cyclopropyl, cyclopentyl, cyclohexyl, cyclopropylmethyl, adamantly, norbornane, and norbornene. This is also true of groups that include the prefix “alkyl-,” such as alkylcarboxylic acid, alkyl alcohol, alkylcarboxylate, alkylaryl, and the like. Examples of suitable alkylcarboxylic acid groups are methylcarboxylic acid, ethylcarboxylic acid, and the like. Examples of suitable alkylacohols are methylalcohol, ethylalcohol, isopropylalcohol, 2-methylpropan-1-ol, and the like. Examples of suitable alkylcarboxylates are methylcarboxylate, ethylcarboxylate, and the like. Examples of suitable alkyl aryl groups are benzyl, phenylpropyl, and the like.

The term “aryl” as used herein includes carbocyclic aromatic rings or ring systems. Examples of aryl groups include phenyl, naphthyl, biphenyl, fluorenyl and indenyl. The term “heteroaryl” includes aromatic rings or ring systems that contain at least one ring hetero atom (e.g., O, S, N). Suitable heteroaryl groups include furyl, thienyl, pyridyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl, thiazolyl, pyrrolyl, tetrazolyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, benzofuranyl, benzothiophenyl, carbazolyl, benzoxazolyl, pyrimidinyl, benzimidazolyl, quinoxalinyl, benzothiazolyl, naphthyridinyl, isoxazolyl, isothiazolyl, purinyl, quinazolinyl, and so on.

The aryl, and heteroaryl groups can be unsubstituted or substituted by one or more substituents independently selected from the group consisting of alkyl, alkoxy, methylenedioxy, ethylenedioxy, alkylthio, haloalkyl, haoalkoxy, haloalkylthio, halogen, nitro, hydroxy, mercapto, cyano, carboxy, formyl, aryl, aryloxy, arylthio, arylalkoxy, arylalkylthio, heteroaryl, heteroaryloxy, heteroarylalkoxy, heteroarylalkylthio, amino, alkylamino, dialkylamino, heterocyclyl, heterocycloalkyl, alkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, haloalkylcarbonyl, haloalkoxycarbonyl, alkylthiocarbonyl, arylcarbonyl, heteroarylcarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, alkanoyloxy, alkanoylthio, alkanoylamino, arylcarbonyloxy, arylcarbonythio, alkylaminosulfonyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aryldiazinyl, alkylsulfonylamino, arylsulfonylamino, arylalkylsulfonylamino, alkylcarbonylamino, alkenylcarbonylamino, arylcarbonylamino, arylalkylcarbonylamino, arylcarbonylaminoalkyl, heteroarylcarbonylamino, heteroarylalkycarbonylamino, alkylsulfonylamino, alkenylsulfonylamino, arylsulfonylamino, arylalkylsulfonylamino, heteroarylsulfonylamino, heteroarylalkylsulfonylamino, alkylaminocarbonylamino, alkenylaminocarbonylamino, arylaminocarbonylamino, arylalkylaminocarbonylamino, heteroarylaminocarbonylamino, heteroarylalkylaminocarbonylamino and, in the case of heterocyclyl, oxo. If other groups are described as being “substituted” or “optionally substituted,” then those groups can also be substituted by one or more of the above enumerated substituents.

The term “arylalkyl,” as used herein, refers to a group comprising an aryl group attached to the parent molecular moiety through an alkyl group.

The term “carbonyl,” as used herein, refers to “C(═O)”.

The terms “acyl,” “carboxyl group,” or “carbonyl group” are recognized in the art and can include such moieties as can be represented by the general formula:

wherein W is OR^(w), N(R^(w))₂, SR^(w), or R^(w), R^(w) being hydrogen, alkyl, heteroalkyl, alkenyl, heteroalkenyl, alkynyl, heteroalkynyl, aryl, heteroaryl, heterocycle, substituted derivatives thereof, or a salt thereof. For example, when W is O-alkyl, the formula represents an “ester,” and when W is OH, the formula represents a “carboxylic acid.” When W is alkyl, the formula represents a “ketone” group, and when W is hydrogen, the formula represents an “aldehyde” group. Those of ordinary skill in the art will understand the use of such terms.

The terms “heterocycle” and “heterocyclic group” are recognized in the art and refer to 3- to about 10-membered ring structures, such as 3- to about 7-membered rings, whose ring structures include one to four heteroatoms. The heterocycle may include portions which are saturated or unsaturated. In some embodiments, the heterocycle may include two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are “fused rings.” In some embodiments, the heterocycle may be a “bridged” ring, where rings are joined through non-adjacent atoms, e.g., three or more atoms are common to both rings. Each of the rings of the heterocycle may be optionally substituted. Examples of heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring may be substituted at one or more positions with substituents including, for example, halogen, aryl, heteroaryl, alkyl, heteroalkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, CF₃, CN, or the like.

As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds, “permissible” being in the context of the chemical rules of valence known to those of ordinary skill in the art. In some cases, “substituted” may generally refer to replacement of a hydrogen with a substituent as described herein. However, “substituted,” as used herein, does not encompass replacement and/or alteration of a key functional group by which a molecule is identified, e.g., such that the “substituted” functional group becomes, through substitution, a different functional group. For example, a “substituted phenyl” must still comprise the phenyl moiety and cannot be modified by substitution, in this definition, to become, e.g., a heteroaryl group such as pyridine. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic, fused, and bridged substituents of organic compounds. Illustrative substituents include, for example, those described herein. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.

According to some aspects of the invention, the PDE inhibitors described herein include compounds having similar bioactivity as those represented by any one of the formulas of groups NI to NHI, but with different chemotypes. Such compounds can be identified by scaffold hopping. The aim of scaffold hopping is to identify isofunctional molecular structures that have the same bioactivity but significantly different molecular backbones. Several methods of scaffold hopping are available and known to one of skill in the art. These include FEPOPS, DAYLIGHT, MACS and Pipeline Pilot fingerprints.

Methods for treating a PDE-associated disease or condition in an individual are provided. The methods include administering to an individual in need of such treatment an effective amount of a compound or composition (e.g., pharmaceutical composition) described herein to treat the PDE-associated disease or condition in the individual. The individual can be a human or other mammal. In some embodiments the PDE-inhibiting compound, which may be a combination of a PDE4 inhibitor, such as a selective PDE4 inhibitor, and a PDE7 inhibitor, such as a selective PDE7 inhibitor, or a combination/dual PDE4/7 inhibitor, is linked to a targeting molecule. In some embodiments the PDE-inhibiting compound is administered prophylactically to a person at risk of developing a PDE-associated disease or disorder.

PDE4 inhibitors, such as selective PDE4 inhibitors (PDE4A, PDE4B) and/or PDE7 inhibitor, such as selective PDE7 inhibitors, and/or dual PDE4/7 inhibitor compounds or pharmaceutical compositions comprising one or more inhibitor and methods described herein, are useful in the treatment (including prevention, partial alleviation or cure) of disorders, which include, but are not limited to, disorders such as: transplant rejection (such as organ transplant, acute transplant, xenotransplant or heterograft or homograft such as is employed in burn treatment); protection from ischemic or reperfusion injury such as ischemic or reperfusion injury incurred during organ transplantation, myocardial infarction, stroke or other causes; transplantation tolerance induction; arthritis (such as rheumatoid arthritis, psoriatic arthritis or osteoarthritis); multiple sclerosis; respiratory and pulmonary diseases including but not limited to asthma, exercise induced asthma, chronic obstructive pulmonary disease (COPD), emphysema, bronchitis, and acute respiratory distress syndrome (ARDS); inflammatory bowel disease, including ulcerative colitis and Crohn's disease; lupus (systemic lupus erythematosis); graft vs. host disease; T-cell mediated hypersensitivity diseases, including contact hypersensitivity, delayed-type hypersensitivity, and gluten-sensitive enteropathy (Celiac disease); psoriasis; contact dermatitis (including that due to poison ivy); Hashimoto's thyroiditis; Sjogren's syndrome; Autoimmune Hyperthyroidism, such as Graves' Disease; Addison's disease (autoimmune disease of the adrenal glands); Autoimmune polyglandular disease (also known as autoimmune polyglandular syndrome); autoimmune alopecia; pernicious anemia; vitiligo; autoimmune hypopituatarism; Guillain-Barre syndrome; other autoimmune diseases; glomerulonephritis; serum sickness; uticaria; allergic diseases such as respiratory allergies (e.g., asthma, hayfever, allergic rhinitis) or skin allergies; scleracierma; mycosis fungoides; acute inflammatory and respiratory responses (such as acute respiratory distress syndrome and ischemia/reperfusion injury); dermatomyositis; alopecia areata; chronic actinic dermatitis; eczema; Behcet's disease; Pustulosis palmoplanteris; Pyoderma gangrenum; Sezary's syndrome; atopic dermatitis; systemic sclerosis; and morphea, and cancer.

Other examples of diseases and disorders associated with cAMP PDE activity and/or abnormal cAMP or cGMP levels include, but are not limited to neurodegenerative disorders, penile erectile dysfunction, anxiety, depression, Alzheimer's disease, Parkinson's disease, Huntington's disease, schizophrenia, psychosis, sepsis, renal disease, memory loss, chronic lymphocytic leukemia, prostate cancer, thyroid disease, male hypogonadism, cardiac disease, diabetes, obesity, osteoporosis, and cystic fibrosis.

DEFINITIONS

A “cyclic AMP phosphodiesterase” or “cAMP PDE” as used herein refers to an enzyme from any biological source which hydrolyzes the substrate 3′,5′-cyclic adenosine monophosphate to yield 5′-adenosine monophosphate. A cAMP PDE may also hydrolyze other substrates, such as 3′,5′-cyclic guanosine monophosphate (cGMP); the enzyme need not have a complete or even a preferential specificity for cAMP. A cAMP PDE of the presently disclosed embodiments can also be a fragment, a mutant, or a post-translationally modified variant of a naturally occurring PDE.

Examples of cAMP PDEs that specifically hydrolyze the substrate 3′,5′-cyclic adenosine monophosphate to yield 5′-adenosine monophosphate and do not hydrolyze 3′,5′-cyclic guanosine monophosphate include: PDE4A, PDE4B, PDE4C, PDE4D, PDE7A, PDE7B, PDE8A, and PDE8B. Examples of cAMP PDEs that hydrolyze the substrate 3′,5′-cyclic adenosine monophosphate to yield 5′-adenosine monophosphate and also hydrolyze 3′,5′-cyclic guanosine monophosphate to yield 5′-guanosine monophosphate include: PDE1A, PDE1B, PDE1C, PDE2A, PDE3A, PDE3B, PDE10A, or PDE11A. It will be understood by those of ordinary skill in the art that the PDEs useful in cells and assays of the invention include PDEs listed herein, and also include splice variants of the PDE families. The identities and sequences of splice variants of PDE families are known and/or are readily identifiable by those of ordinary skill in the art. For example, although not intended to be limiting, PDE4A1 and PDE4A5 are both splice variants of PDE4A, thus the listing of PDE4A herein is understood to include PDE4A1 and PDE4A5. Thus, the invention encompasses the use of splice variants of the PDE families provided herein in cells and assays of the invention. Those of ordinary skill in the art will understand that an exogenous PDE that may be included in a yeast cell of the invention can be from any PDE family listed herein, and that the PDE family members include PDEs provided herein and splice variants thereof.

A “recombinant yeast cell” or “recombinant fission yeast cell” as used herein is a yeast cell into which a foreign nucleic acid (not originating from or identical to a nucleic acid of the same species) has been incorporated by any available technique of molecular biology. Such a recombinant yeast cell may be representative of a larger number of cells, such as a genetic strain, and any cell or method described or claimed herein in the singular is understood to also encompass the plural. A recombinant yeast cell can be, for example, a yeast cell that has been transformed with the DNA encoding a foreign, e.g. exogenous, cAMP PDE. A recombinant yeast cell which is “lacking endogenous PDE” is one that expresses little or no PDE, i.e., 5%, 2%, 1%, or less of the PDE enzyme activity found in a wild type yeast cell of the same species, unless an exogenous gene encoding a PDE has been added to the cell. An “exogenous PDE” is a PDE whose amino acid sequence is different from a PDE of the yeast species into which it is introduced. Exogenous PDE genes for use in the presently disclosed embodiments, include, for example, any human PDE, any mammalian PDE, non-mammalian PDE, and/or any gene from an organism that encodes a protein with PDE activity.

A “fission yeast” or “fission yeast cell” as used herein refers to a unicellular fungus that divides by medial fission. The fission yeast of the presently disclosed embodiments is a yeast of the genus Schizosaccharomyces; a preferred fission yeast is the species Schizosaccharomyces pombe, including any strain derived therefrom. As used herein the terms, “derived from” or “derived therefrom” mean that a yeast strain has been specifically engineered from an original strain. For example, though not intended to be limiting, a cell that includes a cAMP PDE gene and is derived from Schizosaccharomyces pombe (S. pombe), is a cell originated from an S. pombe cell and the S. pombe cell was specifically engineered to include the cAMP PDE gene.

A “reporter construct” as used herein refers to a nucleic acid construct that can be stably transformed into a fission yeast cell, and generally comprises one or more reporter genes under transcriptional control of a promoter. The one or more reporter genes of a reporter construct serve to provide a “detectable signal” upon expression. The detectable signal is any measurable parameter which evidences, in a qualitative or quantitative way, the expression of the reporter gene product in the host fission yeast cell. Examples of detectable signals of reporter genes suitable for use in the presently disclosed embodiments include protein fluorescence (e.g., the fluorescence emission of green fluorescent protein (GFP), red fluorescent protein (RFP), or yellow fluorescent protein (YFP)) and enzyme activity (e.g., β-galactosidase activity), which are well known in the art. Further suitable detectable signals include, but are not limited to, the turbidity, light scattering, or optical density of a cell suspension (indicative of cell growth resulting from reporter activity), or growth in a particular culture medium (e.g., growth in “high glucose” fission yeast culture medium (glucose concentration of at least 3% wt/vol, preferably about 8% wt/vol), or growth in the presence of 5-fluoro-orotic acid (5FOA) or in the absence of uracil). Moreover, activities of fission yeast cells which are dependent on cAMP levels can be used as a detectable signal to monitor PDE activity. Examples include conjugation and sporulation, which require low cAMP levels to occur; higher levels due to PDE inhibition or the absence of a PDE gene would inhibit such processes.

In methods and cells of the invention, a detectable signal may be compared to a control detectable signal. As used herein, a “control detectable signal” is a signal detected in a cell or cell population that is substantially equivalent to the cell or population under equivalent assay conditions, except that a parameter being tested for its effect of PDE activity, for example, a modulating compound (e.g., a test compound), or a cDNA library, is not present in the assay conditions of the control cell or population. A non-limiting example is an assay to identify a modulator of PDE, recombinant yeast cells of the invention may be contacted with a test compound and a detectable signal measured in the cells. A control detectable signal may be the detectable signal generated in a control population of cells that is substantially equivalent (e.g., recombinant with the same genetic characteristics as the test cells) and under essentially the same assay conditions, but the control cells are not contacted with the test compound. Thus, by comparing a detectable signal in cells contacted with the test compound to the detectable signal in cells not contacted with the test compound, differences in the responses of the two populations can be determined. Differences between the test and control, (increases or decreases), are indicative of a modulatory effect of the test compound on the PDE activity. A control detectable signal may be an established value based on previous tests, or may be a signal detected in assays run in parallel with a test assay. Those of ordinary skill in the art will understand and will be able to establish control values, use control values, and compare test with control values using only routine methods.

The promoter determines the transcription of the reporter gene and therefore determines the condition in the cell which is reported as a detectable signal. The promoter can be derived from fission yeast or from another organism. A promoter controls expression of a gene if it is “operably linked” to the gene, which requires that the promoter sequence be situated upstream of the start codon and the open reading frame of the nucleic acid that encodes the reporter protein. In some embodiments, the promoter is “constitutive,” meaning that the gene it controls is continuously expressed. Other promoters provide expression of the gene only when induced by an inducer or certain cell conditions, e.g., low glucose concentration. Promoters suitable for use in the presently disclosed embodiments include, but are not limited to, a constitutive promoter, a PDE promoter, a fission yeast fbp1 promoter, a viral SV40 promoter, and a fission yeast his7 promoter.

The readout for PDE activity is a detectable signal which is sensitive to a change in intracellular cAMP concentration. The terms “cAMP concentration” and “cAMP level” are used interchangeably herein. A level or a concentration of cAMP in a cell can be expressed either in true concentration units (e.g., moles per liter) or in terms of an amount of cAMP per mg of cell protein (e.g., pmol cAMP per mg cell protein); a measurement of cAMP amount on a protein basis can be converted to true concentration units by dividing by cell volume (e.g., in μL per mg protein). In one embodiment, sensitivity of the reporter construct to cAMP is provided through the use of an fbp1 promoter, which is repressed by cAMP-dependent protein kinase (PKA) when cAMP levels rise above approximately 3.5 pmol/mg protein. Other promoters which result in cAMP-dependent reporter gene expression can also be used, such as a git3 or an AC (adenylate cyclase) promoter. As used herein, the phrase “a change in intracellular cAMP concentration” refers to any change in cAMP which produces a detectable signal as a result of reporter gene expression. The “steady-state cAMP concentration” is the concentration of cAMP in a cell prior to the addition of a candidate inhibitor or activator of PDE. Thus, the steady-state cAMP concentration of a given cell or strain can vary depending upon the nature of the experiment (type of culture medium, concentration of glucose, and genetic background). Cyclic AMP levels can be determined by radioimmunoassay, ELISA, or by another method known in the art.

As used herein, the term “5FOA resistant growth” or “growth in the presence of 5FOA” refers to the ability of a fission yeast cell that possesses an fbp1-ura4 fusion reporter gene to grow in the presence of about 0.2 to 1.0 gram/liter, preferably 0.4 gram/liter, 5FOA. Such growth requires a low level of Ura4 activity, which results from a high level of cAMP (e.g., more than 3.5 pmol/mg protein), and corresponds to strong inhibition of PDE. Thus, the greater the amount of 5FOA resistant growth by a fission yeast that possesses an fbp1-ura4 fusion reporter gene, the greater is the extent of PDE inhibition. The amount of growth can be determined after any time interval of exposure to a candidate inhibitor or activator, such that a significant change (e.g., in number of cells, density of cells, cell protein, optical density, light scattering, turbidity, or reporter gene fluorescence) can be experimentally determined. In some embodiments, the amount of growth is determined at about 16 to 24 or about 24 to 48 hours or more following addition of the candidate inhibitor or activator.

“Growth in the absence of uracil” as used herein refers to growth of a fission yeast cell that possesses an fbp1-ura4 fusion reporter gene when cAMP levels are low due to a high PDE activity. Low cAMP levels do not support repression of the fbp1-ura4 reporter construct, such that Ura4 activity is high and cell growth is less dependent on uracil in the medium.

A fission yeast cell that “lacks endogenous ura4 activity” is a cell that expresses little or no ura4 gene product (OMP decarboxylase) from the ura4+ genetic locus, e.g., a cell whose OMP decarboxylase activity is 5%, 2%, 1%, or less compared to a wild type fission yeast cell. A “chemical modulator” of PDE as used herein is a small molecule modulator, i.e., any chemical of less than about 2500 daltons molecular weight which alters the rate of a PDE reaction by at least 5%. A chemical modulator may be a cAMP PDE inhibitor or may be a cAMP PDE activator. A cAMP PDE inhibitor is a modulator that reduces the rate of a PDE reaction by at least 5% and a cAMP PDE activator is a modulator increases the rate of a PDE reaction by at least 5%.

DESCRIPTION

Fission yeast cells can be genetically modified and used as a screening tool to identify inhibitors and activators of PDE. Fission yeast contain only a single PDE gene. If that gene is replaced by a target PDE gene from an exogenous source, and if the appropriate reporter construct or constructs are introduced, the recombinant yeast cells can provide a rapid readout of their intracellular cAMP concentration, which is a measure of PDE activity. Further, the genetic background of the fission yeast cells can be selected to enhance the sensitivity of detecting changes in cAMP level by altering PDE activity. The cells of the presently disclosed embodiments can be further modified by transformation with a cDNA library from a desired cell or tissue source, thereby allowing identification of biological inhibitors and activators of PDE that can be used as novel targets in high throughput drug screens to identify compounds that alter cAMP metabolism.

Recombinant yeast strains have been prepared in which the S. pombe PDE gene was replaced with a target cAMP PDE gene. Such recombinant yeast strains can be used to screen for chemical or biological modulators of the target cAMP PDE activity. Recombinant yeast strains have been prepared using standard yeast manipulations of the genomic DNA to replace the yeast cgs2⁺ gene with that of a mammalian or pathogen cAMP PDE gene. In some embodiments, the cgs2⁺ gene was initially replaced with the ura4⁺ gene. Next, the target cAMP PDE gene was amplified by PCR using oligonucleotides that possess homology to the cgs2 locus. Cells in which this PCR product has replaced the ura4⁺ gene at cgs2 were selected for on 5FOA-containing plates and confirmed by PCR analysis.

The ura4 gene encodes OMP decarboxylase, which is required for uracil biosynthesis and for sensitivity to the pyrimidine analog 5-fluoro-orotic acid (5FOA). Thus, the fbp1-ura4 fusion may be used as either a selectable or a counterselectable marker, making it extremely useful in genetic screens for mutations or clones that increase or decrease fbp1 transcription. The lacZ gene encodes β-galactosidase, which allows its use in sensitive and rapid assays of expression from the fbp1 promoter that are consistent with direct examination of fbp1⁺ mRNA levels. The fbp1-lacZ fusion disrupts ura4 so that all Ura4 activity in these cells comes from the fbp1-ura4 fusion. Strains carrying these fusions were assessed for their ability to regulate fbp1 transcription. Strains that glucose-repress fbp1-ura4 transcription cannot form single colonies on a glucose-rich medium lacking uracil, but grow on a glucose-rich medium containing 5FOA. Strains that fail to glucose-repress fbp1-ura4 form Ura⁺ colonies on a glucose-rich medium lacking uracil. To generalize, strains that are Ura⁺ and 5FOA-sensitive have reduced cAMP levels (either basal or glucose-stimulated) as compared with wild type strains, which are Ura⁻ and 5FOA-resistant.

Recombinant yeast strains of the invention may be used in high-throughput screening for cAMP PDE inhibitors by looking for compounds that confer 5FOA-resistant growth. Conversely, cAMP PDE activators can be identified using the strains and are identified as compounds that confer Ura⁺ growth in strains grown in the presence of enough cAMP to normally prevent growth in SC-ura or EMM-ura medium. In addition, a mammalian cDNA library, such as a human cDNA library, constructed in a fission yeast plasmid expression vector is used to screen for biological modulators of the target PDE. Such modulators are the target of subsequent drug screens and may represent an entirely novel drug target. The advantage of this class of drug target is that it may be expressed in a subset of tissues while the PDE may be expressed in a wider range of cell types. As such, targeting the modulator may limit the effect on PDE activity to the desired cells and reduce side effects relative to drugs that directly target the PDE in all cells in which it is expressed. For example, PDE4 inhibitors produce an emetic response. This response may be due to the inhibition of a particular PDE4 enzyme in the brain. Therefore, PDE4 inhibitors that are specific to either individual PDE4 genes (A, B, C, or D) or even to specific splice variants (4A5, but not 4A1) may be therapeutically useful without producing an emetic response. This, specific inhibitors to PDE4 may be advantageously used for preparing a cAMP PDE modulator as a therapeutic that has minimal negative side-effect.

cAMP Signaling And fbp1 Transcriptional Regulation in Fission Yeast

Both the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae produce cAMP signals in response to glucose detection²⁻⁸. In both yeasts, the increase in cAMP levels is due to the activation of adenylate cyclase, while feedback regulation to limit the cAMP response is, in part, a function of PDE activity⁹⁻¹¹. Studies from a number of labs working in both yeasts have shown that the two signaling pathways share many features; however many important distinctions can be made as well. Most importantly, the S. pombe pathway appears to have a single input in which glucose detection is carried out by the Git3 GPCR that then activates the Gpa2 Gα of the Gpa2-Git5-Git11 heterotrimeric G protein¹²⁻¹⁶. In contrast, the cAMP response in budding yeast involves both the GPCR Gpr1 and the Gpa2 Gα, and a pair of Ras proteins along with the Cdc25 guanine nucleotide exchange factor. In addition, an internal glucose signaling mechanism involving glucose-6-phosphate formation is required for S. cerevisiae cAMP signaling⁸. Thus, the S. pombe cAMP signaling pathway appears to be significantly less complex than that of S. cerevisiae.

Most of the genes that act in the S. pombe cAMP pathway were identified by mutations that inhibit glucose repression of transcription of the fbp1 gene that encodes the gluconeogenic enzyme fructose-1,6-bisphosphate¹⁷. The presently disclosed embodiments employ fbp1-driven reporters that allow for the identification of mutations that alter cAMP levels in the cell. Along with genes required for generating a cAMP signal, which activates PKA, negative regulators of PKA were identified by mutations that suppress the temperature-sensitive growth of a pat1-112 mutant strain¹⁸. The cgs1 gene encodes the regulatory subunit of PKA, while the cgs2 gene encodes the only PDE in S. pombe. Using the fbp1-driven reporters, mutations were identified in cgs1 in a genetic screen for suppressors of an adenylate cyclase deletion allele¹⁹, and mutations in cgs2 in a genetic screen for suppressors of an activation-defective form of adenylate cyclase¹⁰. Thus, a system involving transcriptional regulation of fbp1 is capable of identifying mutations that either reduce or increase PKA activity in the cell.

Recombinant Fission Yeast Containing Reporter Constructs

Translational fusions carrying the fbp1 promoter fused to the S. pombe ura4 and the E. coli lacZ reporter genes can be used to monitor the yeast cell's ability to detect glucose. Additional reporter genes can be used in methods and cells of the invention, including, but not limited to: genes that encode fluorescent proteins and other biosynthetic pathway genes such as his3²⁰. These constructs can be integrated in single copy into the S. pombe genome, creating stable reporters of fbp1 transcription¹⁷. The ura4 gene encodes OMP decarboxylase, which is required for uracil biosynthesis and for sensitivity to the pyrimidine analog 5-fluoro-orotic acid (5FOA). Thus, the fbp1-ura4 fusion acts as a selectable or counterselectable marker, making it extremely useful in genetic screens for mutations or clones that increase or decrease fbp1 transcription. The fbp1-ura4 fusion, for example, can be inserted in single copy into the S. pombe genome at the fbp1 locus and disrupting the wild type fbp1 gene. The lacZ gene encodes β-galactosidase, allowing sensitive and rapid assays of expression from the fbp1 promoter that are consistent with direct examination of fbp1⁺ mRNA levels. The fbp1-lacZ fusion, for example, can be inserted in single copy into the S. pombe genome at the ura4 locus so as to disrupt the wild type ura4 gene, such that all Ura4 enzyme activity in these cells comes from the fbp1-ura4 fusion.

Strains carrying these fusions can be easily assessed for their ability to regulate fbp1 transcription. Strains that glucose-repress fbp1-ura4 transcription cannot form single colonies on a glucose-rich medium lacking uracil because high glucose inhibits OMP decarboxylase expression, thereby reducing uracil biosynthesis. The same strains grow on a glucose-rich medium containing 5FOA because ura4 expression is required for 5FOA sensitivity. Strains that fail to glucose-repress fbp1-ura4 form Ura⁺ colonies on a glucose-rich medium lacking uracil.

In some embodiments, the recombinant fission yeast cell has only a single reporter construct, such as the fbp1-ura4 fusion construct, which can be employed to detect alterations of cAMP level in the cell, and thus inhibition or activation of PDE. Glucose repression of fbp1 is cAMP dependent. High glucose concentrations stimulate adenylate cyclase activity and therefore raise cAMP levels, which stimulate cAMP-dependent protein kinase (PKA) activity. Elevated PKA activity in turn leads to fbp1 repression. Therefore, with the appropriate genetic background providing the appropriate cAMP levels, the growth phenotype of a recombinant fission yeast cell containing the fbp1-ura4 fusion construct can be used to monitor changes in PDE activity. Inhibiting PDE activity will raise cAMP levels, and in a cell possessing the fbp1-ura4 construct inhibiting PDE activity will result in greater glucose-induced repression of Ura4 activity. One consequence of reduced Ura4 activity is loss of 5FOA sensitivity. Thus, in one embodiment, a recombinant fission yeast cell containing a fbp1-ura4 fusion construct is used to identify chemical inhibitors of PDE. When grown in the presence of a test compound which is an inhibitor of PDE, the yeast cell loses 5FOA sensitivity, and therefore grows in the presence of 5FOA when treated with the test compound, but does not grow in 5FOA containing medium in the absence of the test compound.

In other embodiments, the fission yeast cell also has incorporated into its genome a second construct, such as the fbp1-lacZ fusion construct. If the fbp1 promoter is used for both constructs, this permits quantitative monitoring of fbp1+ expression through measurement of β-galactosidase activity. Thus, in one embodiment, a recombinant fission yeast cell contains both an fbp1-ura4 fusion construct and an fbp1-lacZ fusion construct. The level of inhibition of PDE by a test compound can be monitored quantitatively by measuring β-galactosidase activity in the presence of the test compound. The greater the inhibition of PDE, the higher will be the cAMP level in the cell, and consequently, due to cAMP-dependent repression of the fbp1-lacZ construct, the lower will be the β-galactosidase activity. In one embodiment, cells are preincubated, e.g., overnight, in medium containing 1-5 mM cAMP to repress transcription of an fbp1-lacZ reporter construct from the fbp1 promoter and consequently repress β-galactosidase activity. Cyclic AMP then can be washed out by transferring the cells to medium without cAMP at time 0. Washout of cAMP stimulates expression of β-galactosidase to an extent depending on the cellular machinery controlling cAMP levels, including PDE activity.

Alternatively, in a cell possessing both the fbp1-ura4 and fbp1-lacZ constructs, the fbp1 promoter can be used for the ura4 fusion, while a constitutive promoter (e.g., the his7 promoter) can be used to drive a fluorescent protein fusion. In this way, fluorescence can be used to quantitate cell growth. Thus, in another embodiment, a recombinant fission yeast cell contains an fbp1-ura4 fusion construct driven by an fbp1 promoter and an fbp1-lacZ fusion construct driven by a constitutive promoter. The cell can be used to identify an inhibitor of PDE and to quantitate the degree of inhibition. The growth phenotype of the cell can be used to identify test compounds that inhibit PDE; for example, when grown in the presence of a test compound that inhibits PDE, the growth phenotype can switch from 5FOA sensitive to 5FOA tolerant. The amount of growth can be quantified using the fluorescence emission of a fluorescent reporter protein. For example, the greater the amount of fluorescence when grown in the presence of 5FOA, the greater the extent of PDE inhibition by the test compound.

Mutations that Modify cAMP Levels in Fission Yeast

In general, mutations have been identified in nine git genes (git=glucose insensitive transcription) required for glucose repression in fission yeast¹⁷. The increase in fbp1-ura4 expression in git⁻ strains confers a 5FOA-sensitive phenotype that is suppressed by clones carrying the wild type copy of the defective git gene in the host strain or a multicopy suppressor^(13, 14, 16, 19, 21-23). The gene git2 (cyr1) encodes adenylate cyclase, and git6 (pka1) encodes the catalytic subunit of protein kinase A (PKA). Moreover, git1, git3, git5, git7, git8 git10, and git11 are all required for adenylate cyclase activation. Some “upstream” git genes encode a GPCR (git3) and its cognate G protein composed of the Gpa2 Gα, the Git5 Gβ, and the Git11 Gγ. The role of these four genes is to activate the Gpa2 Gα, as mutational activation of Gpa2 suppresses deletions of the other three genes. Since Git1, Git7, and Git10 are still required for glucose repression in a strain expressing an “activated” Gpa2, these proteins may act independently of the G protein or are required for Gpa2 activation of adenylate cyclase. In general, strains that are Ura⁺ and 5FOA-sensitive have reduced cAMP levels (either basal or glucose-stimulated) as compared with wild type strains (see also Table 1, FIG. 1, and Example 1).

While strains that have increased PKA activity are defective in fbp1-ura4 transcription, they largely resemble wild type strains, as it is only under glucose-starvation conditions that a defect in fbp1 transcription is evident. However, by starting with strains with reduced cAMP levels and thus elevated fbp1 expression, mutations have been identified in genes that reduce fbp1-ura4 expression, conferring 5FOA-resistant growth upon the originally 5FOA-sensitive mutant strain. The cgs⁺ gene, encoding the PKA regulatory subunit, was identified in a screen for suppressors of an adenylate cyclase deletion^(18, 19). Strains carrying cgs1 mutations fail to express fbp1 even when cAMP levels are high. The cgs2⁺ gene, encoding the only PDE gene in S. pombe, was identified in a screen for suppressors of a catalytically active form of adenylate cyclase that fails to be stimulated by glucose^(19, 24). Three different mutant alleles of cgs2⁺ have been identified. These mutations reduce PDE activity to different levels and lead to an increase in cAMP levels that is dependent upon the function of adenylate cyclase (Table 1, FIG. 1). A genetic screen has been carried out for activated alleles of the gpa2 Gα gene that bypass the requirement for the Gβγ dimer or Git3 GPCR. These alleles, along with the gpa2^(R176H) GTPase deficient allele, elevate cAMP signaling by raising cAMP levels in the cells (Table 1).

In some embodiments, the recombinant fission yeast cell is a pap1Δ cell. In a pap1α cell, the pap1⁺ gene has been deleted. The deletion of the pap1⁺ gene is not essential for high throughput screening, however it may make the cells more sensitive to both 5FOA and to drug treatment. This pap1⁺ gene encodes a transcriptional activator that regulates the expression of ABC transporter genes. Loss of this gene may allow compounds to accumulate in S. pombe. In certain embodiments, a cell of the invention is a pap1⁺ cell, and therefore does not have the pap1⁺ gene deletion.

Introduction of Exogenous PDE Genes

Recombinant strains of fission yeast can be prepared in which the S. pombe PDE gene is replaced with an exogenous PDE gene to be used for screening to identify chemical or biological modulators of an exogenous PDE activity. Standard yeast manipulations of the genomic DNA, which are well known in the art, can be employed to replace the cgs2⁺ gene with that of an exogenous, e.g., a mammalian or protozoan, PDE gene (or to knock out the cgs2⁺ gene and introduce an exogenous PDE at another site). Typically, this is done in two steps. First, a construct expressing both a selectable marker and a counterselectable marker is introduced by homologous recombination at the cgs2⁺ site, and cells are selected for expression of the marker. These cells will have lost Cgs2 expression and therefore have lost endogenous PDE activity. Second, the exogenous PDE gene is exchanged for the construct added in the first step. The counterselectable marker then can be used to isolate cells having the exogenous PDE gene. As an alternative to replacing the marker at the cgs2 genetic locus with the exogenous PDE gene, the exogenous PDE gene can be integrated into a second genetic locus of a cgs2⁻ mutant strain.

In some embodiments, the ura4⁺ gene serves as both the selectable marker and the counterselectable marker. The cgs2⁺ gene is replaced with the ura4⁺ gene by homologous recombination. Cells having incorporated ura4⁺ are selected based on their growth in the absence of uracil. Next, the exogenous PDE gene is amplified by PCR using oligonucleotides that possess homology to the cgs2 locus, and the exogenous PDE replaces ura4⁺ by homologous recombination. Cells in which the PDE gene has replaced the ura4⁺ gene at cgs2 can be selected on 5FOA-containing plates (i.e., cells incorporating the PDE gene are 5FOA-insensitive, but cells retaining ura4⁺ are 5FOA-sensitive). In another embodiment, the selectable marker is the his7⁺ gene and the counterselectable marker is TK (thymidine kinase). In that case, cells containing his7⁺ can be selected based on growth in the absence of histidine, and TK can be counterselected based on TK-induced sensitivity to 5-fluoro-2-dexoyuridine (FUdr)²⁵.

After the cgs2⁺ gene has been inactivated, and an exogenous PDE gene has been introduced, the resulting yeast cell can be crossed with a yeast cell that contains a reporter construct by standard genetic crosses. The reporter construct encodes a reporter gene whose expression reflects cAMP levels in the cell. For example the reporter construct can be an fbp1-ura4 fusion reporter construct. A second reporter construct, e.g., an fbp1-lacZ fusion construct, can also be added by crossing. For these crosses, a fission yeast background strain can be selected which has a sufficiently high level of adenylate cyclase activation such that the exogenous PDE activity can support a 5FOA-sensitive growth behavior. For example, if the exogenous PDE activity is similar to that of the normal yeast PDE, even a weak mutation, such as the loss of git11 (see Table 1), would confer 5FOA-sensitive growth. If, however, the exogenous PDE activity is relatively low, a greater defect in the cAMP pathway, such as the loss of the git3 or gpa2 genes (Table 1), could be required to confer 5FOA-sensitive growth. Should the PDE be so weak that even loss of the gpa2 gene does not confer 5FOA-sensitivity, a deletion of the adenylate cyclase gene could be incorporated and endogenous cAMP production could be replaced by exogenous cAMP addition to create the conditions needed for a PDE inhibitor screen. If the PDE is very active, it may confer 5FOA-sensitivity even in a wild type background. In this case, activated forms of the gpa2 gene (Welton and Hoffman, supra) can be introduced to increase cAMP production, in order to make the cells more sensitive to changes in the PDE activity.

Screening Assays

The recombinant fission yeast cells described above can be used in high throughput chemical screens to identify PDE inhibitors that confer 5FOA-resistant growth.

Screening assays can be adapted from the use of solid media to working in liquid media in microtiter plates suitable for chemical library screening. PDE inhibitors would confer increased optical density in the affected wells due to cell growth, along with increased fluorescence from a constitutively expressed fluorescent protein reporter. Preferably, a positive growth screen is used, such as growth in the absence of uracil or in the presence of 5FOA, so that compounds that are toxic to the cells or impermeable will not yield a positive result and can be avoided.

Test compounds or agents to be screened can be naturally occurring or synthetic molecules. The activity of the compounds can be known or unknown. Test compounds can be obtained from natural sources, such as, for example, marine microorganisms, algae, plants, fungi, etc. Test compounds can include, for example, pharmaceuticals, therapeutics, environmental, agricultural, or industrial agents, pollutants, cosmeceuticals, drugs, organic compounds, lipids, fatty acids, steroids, glucocorticoids, antibiotics, peptides, proteins, sugars, carbohydrates, chimeric molecules, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

Collections of compounds known as libraries can be used for screening. Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from governmental or private sources or can be produced readily. Alternatively, agents to be assayed can be from combinatorial libraries of agents, including peptides or small molecules, or from existing repertories of chemical compounds synthesized in industry, e.g., by the chemical, pharmaceutical, environmental, agricultural, marine, drug, and biotechnological industries. Preparation of combinatorial chemical libraries is well known to those of skill in the art. Compounds that can be synthesized for combinatorial libraries include polypeptides, proteins, nucleic acids, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines, and oligocarbamates. Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville, Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.). Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries.

Screening may also be directed to known pharmacologically active compounds and analogs thereof. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, coalkylation, esterification, amidification, etc. to produce structural analogs. New potential test agents may also be created using methods such as rational drug design or computer modeling.

As described above, compounds that may be assayed according to the methods of the presently disclosed embodiments encompass numerous chemical classes. For example, organic molecules, preferably small organic compounds having a molecular weight less than about 2,500 daltons, are a type of compound for use in the methods of the presently disclosed embodiments.

In the methods of the presently disclosed embodiments, each test compound, or a composition comprising the test compound, is brought into contact with a cell or plurality of cells in a manner such that the test compound is capable of exerting activity on at least a substantial portion of, if not all of, the individual cells. By substantial portion is meant at least 75%, usually at least 80%, and in many embodiments 90% or 95% or higher percentage of the cells are exposed to the test compound. Generally, a cell is contacted with a test compound in a manner such that the compound is internalized by the cells. For example, the test compound can be added into a growth medium or incubation solution in which the cell is suspended or upon which the cell is growing. Compounds are generally screened at a concentration in the range expected for them to be effective, e.g., as PDE inhibitors, or somewhat above that concentration. Any concentration below 1 mM may be chosen, but screening assays are often conducted with test compounds at about 7 μM, about 20 μM, or about 50 μM.

After high throughput screening (primary screening), several candidate inhibitors or activators of PDE will have been identified. These inhibitor or activator compounds can be further tested using a secondary screen, such as an in vitro assay wherein the compounds are tested using purified PDE under controlled conditions. The secondary screen can further identify the most desirable compounds, for example those with the highest potency (e.g., lowest K₁ value for an inhibitor compound).

PDE-Modulating Compounds

Methods of the invention involve the administration of compounds that modulate the activity of PDEs. In certain embodiments the hydrolysis of the substrate 3′,5′-cyclic adenosine monophosphate (cAMP) to yield 5′-adenosine monophosphate or the hydrolysis of another substrate, such as 3′,5′-cyclic guanosine monophosphate (cGMP) is modulated. Compositions of the invention include compounds that modulate or inhibit PDE activity in vitro or in vivo, in cells, tissues, or subjects, which may be mammals or humans. As used herein, the term “PDE-inhibiting compounds” means compounds that reduce PDE hydrolysis of its substrate, which in some embodiments may be cAMP and in certain embodiments may be cGMP. The methods of the invention, in some aspects, involve the administration of a PDE-inhibiting compound and are useful to reduce or prevent adverse effects that are associated with abnormal levels of PDE substrates such as cAMP and/or cGMP, for example, cell death and/or damage or disease.

As used herein, the term “PDE-associated disease or disorder” includes, but is not limited to diseases and disorders in which there is abnormal PDE activity and/or abnormal levels of a substrate of a PDE, such as cAMP and/or cGMP. As used herein, the term “PDE activity” means PDE-mediated hydrolysis of a substrate such as cAMP or cGMP. An abnormal level of PDE activity and/or an abnormal level of a substrate may be a level that is higher than a normal level or may be a level that is lower than a normal level, wherein a “normal” level is the level in a subject who does not have a disease or disorder associated with PDE activity or with an abnormal level of cAMP or cGMP. Disease or disorders that may be associated with PDE activity and abnormal cAMP or cGMP levels, and which may benefit from treatment according to the methods described herein using compounds of the invention, are: transplant rejection (such as organ transplant, acute transplant, xenotransplant or heterograft or homograft such as is employed in burn treatment); protection from ischemic or reperfusion injury such as ischemic or reperfusion injury incurred during organ transplantation, myocardial infarction, stroke or other causes; transplantation tolerance induction; arthritis (such as rheumatoid arthritis, psoriatic arthritis or osteoarthritis); multiple sclerosis; respiratory and pulmonary diseases including but not limited to asthma, exercise induced asthma, chronic obstructive pulmonary disease (COPD), emphysema, bronchitis, and acute respiratory distress syndrome (ARDS); inflammatory bowel disease, including ulcerative colitis and Crohn's disease; lupus (systemic lupus erythematosis); graft vs. host disease; T-cell mediated hypersensitivity diseases, including contact hypersensitivity, delayed-type hypersensitivity, and gluten-sensitive enteropathy (Celiac disease); psoriasis; contact dermatitis (including that due to poison ivy); Hashimoto's thyroiditis; Sjogren's syndrome; Autoimmune Hyperthyroidism, such as Graves' Disease; Addison's disease (autoimmune disease of the adrenal glands); Autoimmune polyglandular disease (also known as autoimmune polyglandular syndrome); autoimmune alopecia; pernicious anemia; vitiligo; autoimmune hypopituatarism; Guillain-Barre syndrome; other autoimmune diseases; glomerulonephritis; serum sickness; uticaria; allergic diseases such as respiratory allergies (e.g., asthma, hayfever, allergic rhinitis) or skin allergies; scleracierma; mycosis fungoides; acute inflammatory and respiratory responses (such as acute respiratory distress syndrome and ischemia/reperfusion injury); dermatomyositis; alopecia areata; chronic actinic dermatitis; eczema; Behcet's disease; Pustulosis palmoplanteris; Pyoderma gangrenum; Sezary's syndrome; atopic dermatitis; systemic sclerosis; and morphea, and cancer, but are not so limited.

Other examples of diseases and disorders associated with cAMP PDE activity and/or abnormal cAMP or cGMP levels include, but are not limited to neurodegenerative disorders, penile erectile dysfunction, anxiety, depression, Alzheimer's disease, Parkinson's disease, Huntington's disease, schizophrenia, psychosis, sepsis, renal disease, memory loss, chronic lymphocytic leukemia, prostate cancer, thyroid disease, male hypogonadism, cardiac disease, diabetes, obesity, osteoporosis, and cystic fibrosis.

Deleterious effects seen in these diseases and/or disorders that are triggered by abnormal PDE activity and/or abnormal levels of a substrate of a PDE (e.g., cAMP or cGMP) may be ameliorated by the administration of compounds and/or compositions that modulate PDE activity. The compounds or compositions may comprise for example at least one PDE inhibitor, which may be selective for a PDE family, a specific PDE subfamily, or a specific isoform of a PDE-subfamily member, such as a selective PDE4 inhibitor or a selective PDE7 inhibitor, or a dual PDE7-PDE4 inhibitor.

Compounds of the invention include compounds that modulate PDE activity in the hydrolysis of substrates such as cAMP and cGMP in cells and/or tissues (in a subject), thereby reducing the cell and/or tissue damage and/or clinical manifestations of a PDE-associated disease or disorder. In some embodiments of the invention, the compounds inhibit PDE activity, thus resulting in an increase in levels of cAMP and/or cGMP.

A compound of the invention may be an isolated compound. By “isolated”, it is meant present in sufficient quantity to permit its identification or use according to the procedures described herein. Because an isolated material may be admixed with a carrier in a preparation, such as, for example, for adding to a sample or for analysis, the isolated material may comprise only a small percentage by weight of the preparation.

In some aspects of the invention, one or more of compounds described herein may be administered to a subject that is free of indications for a previously determined use of the compounds. By “free of indications for a previously determined use”, it is meant that the subject does not have symptoms that call for treatment with one or more of the compounds of the invention for a previously determined use of that compound, other than the indication that exists as a result of this invention. As used herein the term “previously determined use” of a compound means the use of the compound that was previously identified. Thus, the previously determined use is not the use of inhibiting PDE activity and/or increasing the level of a PDE substrate such as cAMP and/or cGMP.

Administration and Delivery of PDE Modulating Compounds

Methods of the invention, in some aspects, include administration of a PDE-inhibiting compound that preferentially targets neuronal or vascular cells and/or tissues or other specific cell or tissue types. In addition, the compounds can be specifically targeted to neuronal or vascular tissue or other specific tissue types. The targeting may be done using various delivery methods, including, but not limited to: administration to neuronal or vascular tissue or other specific target tissue, the addition of targeting molecules to direct the compounds of the invention to neuronal or other tissues (e.g. glial cells, nerve cells, vascular cells, etc.). Additional methods to specifically target compounds and compositions of the invention to specific tissues, such as neuronal tissues, vascular tissues, or other types of tissues may also be used with the compounds and compositions of the invention, and are known to those of ordinary skill in the art.

In certain embodiments the invention provides compounds that inhibit PDE activity in cells, tissues, and/or subjects and the use of such compounds to inhibit PDE. PDE inhibitors of the invention, such as selective PDE4 inhibitors or selective PDE7 inhibitors, or a dual PDE7-PDE4 inhibitors, may be used for treatment of cells, tissues, and/or subjects and for research purposes. As used herein, the term “PDE activity” means the hydrolysis of PDE substrate such as cAMP and/or cGMP. It is understood that increased activity of a PDE may result in an abnormally low level of cAMP or cGMP. Also, it will be understood, that for reasons unrelated to the activity of a PDE in a cell, tissue or subject, a level of cAMP and/or cGMP may be below a desirable level (e.g., at an abnormally low level) and methods and compounds of the invention may be used to inhibit PDE activity and thereby increase the level of cAMP and/or cGMP in the cell, tissue, or subject.

PDE-inhibiting compounds of the invention may be administered to a subject to reduce the risk of a PDE-associated disorder. Reducing the risk of a disorder associated with above-normal PDE activity or a associated with abnormally low levels of a substrate of a PDE (e.g., cAMP and/or cGMP), means using treatments and/or medications that include compounds of the invention, such as compounds comprising selective PDE4 inhibitors or selective PDE7 inhibitors, or a dual PDE7-PDE4 inhibitors, to reduce PDE activity levels, therein increasing the subject's levels of the substrate, e.g., cAMP and/or cGMP and thus treating the associated disease or disorder.

As used herein, the term “individual” means any mammal who may be in need of treatment with a PDE-modulating or inhibiting compound of the invention. They include but are not limited to: humans, non-human primates, cats, dogs, sheep, pigs, horses, cows, rodents, such as mice, and rats.

As used herein the term “inhibit” means to reduce the amount of PDE activity to a level or amount that is less than an initial level, which may be a control level of PDE activity and/or PDE substrate hydrolysis. An initial level may be a level in a cell, tissue, or subject not contacted with a PDE-inhibiting compound of the invention. In some cases, the decrease in the level of PDE activity and/or PDE substrate hydrolysis means the level of PDE activity and/or substrate hydrolysis is reduced from an initial level to a level significantly lower than the initial level. In some embodiments, the reduced level may be zero.

A PDE-modulating compound of the invention (e.g., a PDE inhibitor, such as a selective PDE4 inhibitor or a selective PDE7 inhibitor, or a dual PDE7-PDE4 inhibitor) may be used to treat a subject with a PDE-associated disease or disorder. As used herein, the term “treat” includes active treatment of a subject that has a PDE-associated disease or disorder (e.g., a subject diagnosed with such a condition) and also includes prophylactic treatment of a subject who has not yet been diagnosed and/or has not yet developed a PDE-associated disease. Compounds of the invention may administered prophylactically to a subject at risk of a PDE-associated disease or disorder. Determination of a subject at risk for a PDE-associated disease or disorder, and/or the determination of a diagnosis of a PDE-associated disease or disorder in a subject, may be carried out by one of ordinary skill in the art using routine methods.

A PDE-modulating or inhibiting compound of the invention may be delivered to a cell using standard methods known to those of ordinary skill in the art. Various techniques may be employed for introducing PDE-modulating compounds of the invention to cells, depending on whether the compounds are introduced in vitro or in vivo in a host.

When administered, the PDE-modulating compounds (also referred to herein as therapeutic compounds and/or pharmaceutical compounds) of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.

The term “pharmaceutically acceptable” carrier means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The characteristics of the carrier will depend on the route of administration.

The therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may for example, be oral, intravenous, intraperitoneal, intrathecal, intramuscular, intranasal, intracavity, subcutaneous, intradermal, mucosal, transdermal, or transdermal.

The therapeutic compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the compounds into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the therapeutic agent, which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.

Compositions suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets, or lozenges, each containing a predetermined amount of the therapeutic agent. Other compositions include suspensions in aqueous liquors or non-aqueous liquids such as a syrup, an elixir, or an emulsion.

In some embodiments of the invention, a PDE-modulating compound of the invention may be delivered in the form of a delivery complex. The delivery complex may deliver the PDE-modulating compound into any cell type, or may be associated with a molecule for targeting a specific cell type. Examples of delivery complexes include a PDE-modulating compound of the invention associated with: a sterol (e.g., cholesterol), a lipid (e.g., a cationic lipid, virosome or liposome), or a target cell specific binding agent (e.g., an antibody, including but not limited to monoclonal antibodies, or a ligand recognized by target cell specific receptor). Some complexes may be sufficiently stable in vivo to prevent significant uncoupling prior to internalization by the target cell. However, the complex can be cleavable under appropriate conditions within the cell so that the PDE-modulating compound is released in a functional form.

An example of a targeting method, although not intended to be limiting, is the use of liposomes to deliver a PDE-modulating compound of the invention into a cell. Liposomes may be targeted to a particular tissue, such neuronal cells, (e.g. hippocampal cells, etc), or other cell type, by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein. Such proteins include proteins or fragments thereof specific for a particular cell type, antibodies for proteins that undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like.

For certain uses, it may be desirable to target the compound to particular cells, for example specific neuronal cells, including specific tissue cell types, e.g. tissue-specific nervous system cells. In some embodiments, it may be desirable to target a PDE-modulating compound to another cell type, including, but not limited to, cardiac cells, pancreatic cells, vascular cells, etc. In such instances, a vehicle (e.g. a liposome) used for delivering a PDE-modulating compound of the invention to a cell type (e.g. a neuronal cell, vascular cell, etc.) may have a targeting molecule attached thereto that is an antibody specific for a surface membrane polypeptide of the cell type or may have attached thereto a ligand for a receptor on the cell type. Such a targeting molecule can be bound to or incorporated within the PDE-modulating compound delivery vehicle. Where liposomes are employed to deliver a PDE-modulating compound of the invention, proteins that bind to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation for targeting and/or to facilitate uptake.

Liposomes are commercially available from Invitrogen, for example, as LIPOFECTIN™ and LIPOFECTACE™, which are formed of cationic lipids such as N-[1-(2,3 dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Methods for making liposomes are well known in the art and have been described in many publications.

The invention provides a composition of the above-described agents for use as a medicament, methods for preparing the medicament and methods for the sustained release of the medicament in vivo. Delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the therapeutic agent of the invention, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include, but are not limited to, polymer-based systems such as polylactic and polyglycolic acid, poly(lactide-glycolide), copolyoxalates, polyanhydrides, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polycaprolactone. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Nonpolymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di- and tri-glycerides; phospholipids; hydrogel release systems; silastic systems; peptide based systems; wax coatings, compressed tablets using conventional binders and excipients, partially fused implants and the like. Specific examples include, but are not limited to: (a) erosional systems in which the polysaccharide is contained in a form within a matrix, found in U.S. Pat. Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.

In one particular embodiment, the preferred vehicle is a biocompatible microparticle or implant that is suitable for implantation into the mammalian recipient. Exemplary bioerodible implants that are useful in accordance with this method are described in PCT International application no. WO 95/24929, entitled “Polymeric Gene Delivery System”. describes a biocompatible, preferably biodegradable polymeric matrix for containing an exogenous gene under the control of an appropriate promoter. The polymeric matrix is used to achieve sustained release of the exogenous gene in the patient. In accordance with the instant invention, the compound(s) of the invention is encapsulated or dispersed within the biocompatible, preferably biodegradable polymeric matrix disclosed in WO 95/24929. The polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein the compound is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the compound is stored in the core of a polymeric shell). Other forms of the polymeric matrix for containing the compounds of the invention include films, coatings, gels, implants, and stents. The size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix device is implanted. The size of the polymeric matrix device further is selected according to the method of delivery which is to be used. The polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the device is administered to a vascular surface. The matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time.

Both non-biodegradable and biodegradable polymeric matrices can be used to deliver agents and compounds of the invention of the invention to the subject. Biodegradable matrices are preferred. Such polymers may be natural or synthetic polymers. Synthetic polymers are preferred. The polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable. The polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multi-valent ions or other polymers.

In general, the agents and/or compounds of the invention are delivered using the bioerodible implant by way of diffusion, or more preferably, by degradation of the polymeric matrix. Exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone.

Examples of non-biodegradable polymers include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Examples of biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.

Bioadhesive polymers of particular interest include bioerodible hydrogels may include, but are not limited to: polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

Use of a long-term sustained release implant may be particularly suitable for treatment of subjects with an established neurological disorder or other cAMP PDE-associated disease or disorder as well as subjects at risk of developing a such a disease or disorder.

“Long-term” release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and preferably 30-60 days, and most preferably months or years. The implant may be positioned at or near the site of the neurological damage or the area of the brain or nervous system affected by or involved in the neurodegenerative disorder. Long-term release implants may also be used in non-neuronal tissues and organs to allow regional administration of a PDE-modulating compound of the invention. Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the release systems described above.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

PDE inhibitor compounds described herein, include salts, prodrugs and solvates. The term “salt(s)”, as employed herein, denotes acidic and/or basic salts formed with inorganic and/or organic acids and bases and Zwitterions (internal or inner salts) are also included. Also included herein are quaternary ammonium salts such as alkylammonium salts. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred.

Exemplary acid addition salts include acetates (such as those formed with acetic acid or trihaloacetic acid, for example, trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides, hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates (such as those formed with sulfuric acid), sulfonates (such as those mentioned herein), tartrates, thiocyanates, toluenesulfonates, undecanoates, and the like.

Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as benzathines, dicyclohexylamines, hydrabamines, N-methyl-D-glucamines, N-methyl-D-glucamides, t-butyl amines, and salts with amino acids such as arginine, lysine and the like.

Prodrugs and solvates of the compounds of the invention are also contemplated herein. The term “prodrug”, as employed herein, denotes a compound which, upon administration to a subject, undergoes chemical conversion by metabolic or chemical processes to yield a compounds described herein or a salt and/or solvate thereof.

All stereoisomers of the present compounds, including enantiomeric and diastereomeric forms, are contemplated within the scope of this invention. Individual stereoisomers of the compounds of the invention may, for example, be substantially free of other isomers, or may be admixed, for example, as racemates or with all other, or other selected, stereoisomers. The chiral centers of the present invention can have the S or R configuration as defined by the IUPAC 1974 Recommendations.

The preparations of the invention are administered in effective amounts. An effective amount is that amount of a pharmaceutical preparation that alone, or together with further doses, results in the desired response. In the case of treating a disorder or condition that is associated with abnormal PDE activity and/or abnormal levels of cAMP, desired response is reducing the onset, stage or progression of the abnormal PDE activity and/or levels of cAMP and associated effects. This may involve only slowing the progression of the damage temporarily, although more preferably, it involves halting the progression of the damage permanently. An effective amount for treating abnormal PDE activity and/or cAMP levels is that amount that alters (increases or reduces) the amount or level of PDE activity and/or cAMP level, when the cell or subject is a cell or subject with a PDE-associated disease or disorder, with respect to that amount that would occur in the absence of the active compound.

The invention involves, in part, the administration of an effective amount of a PDE-modulating compound of the invention. The PDE-modulating compounds of the invention are administered in effective amounts. Typically effective amounts of a PDE-modulating compound will be determined in clinical trials, establishing an effective dose for a test population versus a control population in a blind study. In some embodiments, an effective amount will be that amount that diminishes or eliminates a PDE-associated disease or disorder and its effects in a cell, tissue, and/or subject. Thus, an effective amount may be the amount that when administered reduces the amount of cell and or tissue damage and/or, cell death from the amount that would occur in the subject or tissue without the administration of a PDE-modulating compound of the invention.

The pharmaceutical compound dosage may be adjusted by the individual physician or veterinarian, particularly in the event of any complication. A therapeutically effective amount typically varies from 0.01 mg/kg to about 1000 mg/kg, preferably from about 0.1 mg/kg to about 200 mg/kg, and most preferably from about 0.2 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or more days. It will be recognized by those of skill in the art that some of the PDE-modulating compounds may have detrimental effects at high amounts. Thus, an effective amount for use in the methods of the invention may be optimized such that the amount administered results in minimal negative side effects and maximum PDE modulation. The amount can be varied in order to reduce negative side effects (dosage can be varied, as can schedule for administration).

The absolute amount will depend upon a variety of factors, including the material selected for administration, whether the administration is in single or multiple doses, and individual subject parameters including age, physical condition, size, weight, and the stage of the disease or disorder. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.

Alternative drug therapies are known to those of ordinary skill in the art and are administered by modes known to those of skill in the art. The drug therapies are administered in amounts that are effective to achieve the physiological goals (to reduce symptoms and damage from a PDE-associated disease or disorder in a subject, e.g. cell damage and/or cell death), in combination with the pharmaceutical compounds of the invention. Thus, it is contemplated that the alternative drug therapies may be administered in amounts which are not capable of preventing or reducing the physiological consequences of the PDE-associated disease and/or disorder when the drug therapies are administered alone, but which are capable of preventing or reducing the physiological consequences of a PDE-associated disease and/or disorder when administered in combination with one or more PDE-modulating compounds of the invention.

Diagnostic tests known to those of ordinary skill in the art may be used to assess the level of PDE activity and/or levels of cAMP in a subject and the effects thereof, and to evaluate a therapeutically effective amount of a pharmaceutical compound administered. Examples of diagnostic tests are set forth below. A first determination of PDE activity, level of cAMP, and/or the effects thereof in a cell and/or tissue may be obtained using one of the methods described herein (or other methods known in the art), and a second, subsequent determination of the level of PDE activity or level of cAMP. A comparison of the PDE activity and/or cAMP level and/or the effects thereof on the subject at the different time points may be used to assess the effectiveness of administration of a pharmaceutical compound of the invention as a prophylactic or an active treatment of the PDE-associated disease or disorder. Family history or prior occurrence of a PDE-associated disease or disorder, even if the PDE-associated disease or disorder is absent in a subject at present, may be an indication for prophylactic intervention by administering a pharmaceutical compound described herein to reduce or prevent abnormal PDE activity and/or abnormal levels of cAMP.

An example of a method of diagnosis of abnormal PDE activity and/or abnormal levels of cAMP that can be performed using standard methods such as, but not limited to: imaging methods, electrophysiological methods, blood tests, and histological methods. Additional methods of diagnosis and assessment of PDE-associated disease or disorders and the resulting cell death or damage are known to those of skill in the art.

In addition to the diagnostic tests described above, clinical features of PDE-associated diseases and/or disorders can be monitored for assessment of PDE activity following onset of a PDE-associated disease or disorder. These features include, but are not limited to: assessment of the presence of cell damage, cell death, neuronal cell lesions, brain lesions, organ lesions, vascular damage, blood abnormalities, sugar processing abnormalities, and behavioral abnormalities. Such assessment can be done with methods known to one of ordinary skill in the art, such as behavioral testing, blood testing, and imaging studies, such as radiologic studies, CT scans, PET scans, etc.

The pharmaceutical compounds of the invention may be administered alone, in combination with each other, and/or in combination with other drug therapies that are administered to subjects with PDE-associated diseases or disorders.

In some embodiments the PDE-inhibiting compound is administered in combination with an additional drug for treating a PDE-associated disease or disorder. For example, selective PDE4 inhibitors or selective PDE7 inhibitors or dual PDE4-PDE7 inhibitor compounds described herein, may be administered alone or in combination with other suitable therapeutic agents useful in treating immune and inflammatory disorders such as: immunosuppressants such as, cyclosporins (e.g., cyclosporin A), anti-IL-1 agents, such as Anakinra, the IL-1 receptor antagonist, CTLA4-Ig, antibodies such as anti-ICAM-3, anti-IL-2 receptor (Anti-Tac), anti-CD45RB, anti-CD2, anti-CD3, anti-CD4, anti-CD80, anti-CD86, monoclonal antibody OKT3. agents blocking the interaction between CD40 and CD154, such as antibodies specific for CD40 and/or CD154 (i.e., CD40L), fusion proteins constructed from CD40 and CD154 (CD40Ig and CD8-CD154), interferon beta, interferon gamma, methotrexate, FK506 (tacrolimus, Prograf), rapamycin (sirolimus or Rapamune) mycophenolate mofetil, leflunomide (Arava), azathioprine and cyclophosphamide, inhibitors, such as nuclear translocation inhibitors, of NF-kappa B function, such as deoxyspergualin (DSG), non-steroidal antiinflammatory drugs (NSAIDs) such as ibuprofen, cyclooxygenase-2 (COX-2) inhibitors such as celecoxib (Celebrex) and rofecoxib (Vioxx), or derivatives thereof, steroids such as prednisone or dexamethasone, gold compounds TNF-.alpha. inhibitors such as tenidap, anti-TNF antibodies or soluble TNF receptor such as etanercept (Enbrel), inhibitors of p-38 kinase such as BIRB-796, RO-3201195, VX-850, and VX-750, beta-2 agonists such as albuterol, levalbuterol (Xopenex), and saltmeterol (Screvent), inhibitors of leukotriene synthesis such as montelukast (Singulair) and zariflukast (Accolate), and anticholinergic agents such as ipratropium bromide (Atrovent). PDE4 inhibitors such as Arofyline, Cilomilast, Roflumilast, C-11294A, CDC-801, BAY-19-8004, Cipamfylline, SCH351591, YM-976, PD-189659, Mesiopram, Pumafentrine, CDC-998, IC-485, and KW-4490, PDE7 inhibitors such as IC242, (Lee, et. al. PDE7A is expressed in human B-lymphocytes and is up-regulated by elevation of intracellular cAMP. Cell Signalling, 14, 277-284, (2002)) and also include compounds disclosed in the following patent documents: WO 0068230, WO 0129049, WO 0132618, WO 0134601, WO 0136425, WO 0174786, WO 0198274, WO 0228847, U.S. Provisional Application Ser. No. 60/287,964, and U.S. Provisional Application Ser. No. 60/355,141 anti-cytokines such as anti-IL-1 mAb or IL-1 receptor agonist, anti-IL-4 or IL-4 receptor fusion proteins and PTK inhibitors such as those disclosed in the following U.S. patents and applications, incorporated herein by reference in their entirety: U.S. Pat. Nos. 6,235,740, 6,239,133, U.S. application Ser. No. 60/065,042, filed Nov. 10, 1997, U.S. application Ser. No. 09/173,413, filed Oct. 15, 1998, and U.S. Pat. No. 5,990,109.

REFERENCES FOR DETAILED DESCRIPTION OF THE INVENTION

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EXAMPLES

The examples below are non-limiting and are merely representative of various aspects and features of the presently disclosed embodiments.

Example 1 Construction of a Recombinant Fission Yeast Strain Capable of Reporting Changes in cAMP Concentration

Translational fusions carrying the fbp1 promoter fused to the S. pombe ura4 and the E. coli lacZ reporter genes were prepared and used to monitor the cell's ability to detect glucose. See Hoffman, C. S. and F. Winston, Genetics, 1990, 124(4): p. 807-16. These constructs were integrated in single copy into the S. pombe genome, creating stable reporters of fbp1 transcription.

Fission yeast strains were spotted onto yeast extract agar supplemented with 2% casamino acids (YEA medium) and grown overnight. PDE activity was then assessed by replica plating the cells onto either YEA medium, synthetic complete (SC) solid medium containing 8% glucose and 0.4 g/L 5-fluorourotic acid (5FOA medium), or SC medium containing 8% glucose with no uracil (SC-Ura medium). For details, including β-galactosidase and cAMP assays, see Wang et al., Genetics, 2005, 171, p. 1523-33. The results are shown in FIG. 1 and Table 1.

The cgs2-s1 and cgs2-s4 PDE gene mutations were isolated based on their ability to confer 5FOA-resistant growth to a strain carrying a mutation that prevented adenylate cyclase stimulation, leaving strains lacking adenylate cyclase 5FOA-sensitive (FIG. 1). In addition, the PDE mutations differentially suppress the loss of the gpa2 gene (Table 1; compare gpa2Δ cgs2-s1 and gpa2Δ cgs2-2), demonstrating that different reductions in PDE activity can be required to confer 5FOA-resistance depending upon the genetic background of the strain. In effect, different mutations that affect the generation of cAMP can be used to “tune” the cells such that their growth behavior reflects the level of PDE activity. See Wang et al., Genetics, 2005, 171(4): p. 1523-33 for description of the mutations.

TABLE 1 Phenotypes associated with fbp1 reporters in different genetic backgrounds. Strain βgal level repressed 5FOA growth basal cAMP level Wild type 10 ++ 3.6 git3Δ (GPCR) 925 − 1.7 gpa2Δ (Gα) 1400 − 2.0 git5Δ (Gβ) 1050 − 3.2 git11Δ (GΔ) 300 − ND gpa2Δ cgs2-s1 480 − ND gpa2Δ cgs2-2 10 ++ ND git3Δ cgs2-s1 30 ++ 4.4 git3Δ cgs2-2 4 ++ 11.6 cgs2-s1 4 ++ 4.1 cgs2-2 7 ++ 13.3 gpa2^(R176H) 5 ++ 6.9

Example 2 Quantification of cAMP Levels Using Recombinant Fission Yeast

Wild type and two mutant strains (git1-1 and git2-7) having reduced cAMP levels were incubated overnight (18-24 hours) in EMM medium containing 5 mM cAMP to repress transcription of an fbp1-lacZ reporter construct from the fbp1 promoter and consequently repress β-galactosidase activity. Cyclic AMP was washed out by transferring the cells to EMM without cAMP at time 0. Washout of cAMP stimulated expression of β-galactosidase to an extent depending on the cellular machinery controlling cAMP levels. The results are shown in FIG. 2. The relative sensitivity of the mutant strains to 5FOA is shown in Table 2. The git1-1 strain, which was considerably more sensitive to 5FOA, yields the highest β-galactosidase activity after washout of cAMP in FIG. 2, demonstrating a semi-quantitative correlation between cAMP metabolism and cell growth in the presence of 5FOA.

TABLE 2 Growth of S. pombe strains in 5FOA-containing medium correlates with effect of mutations on fbp1 expression. Fold increase Genotype −cAMP +cAMP Wild type 146 122 git2-7 10 86 git1-1 3.6 80 The fold increase in cell number is shown following 24 hours growth after transfer to 0.4 g/L 5FOA in the presence or absence of 5 mM cAMP.

Example 3 Use of a Recombinant Fission Yeast for High Throughput Screening for Chemical Inhibitors of PDE

Two 5FOA-sensitive strains are pregrown in the presence of 5 mM cAMP to repress transcription from the fbp1 promoter. Both strains possess the fbp1-ura4 and fbp1-lacZ reporter constructs. The experimental strain also expresses PDE4A1 in place of the yeast PDE. The control strain expresses the endogenous yeast PDE. Each strain is put individually into 384 well microtiter plates in a growth medium that contains 5FOA and 8% glucose, but no exogenous cAMP. These plates are used to screen a chemical library using robots that pin various compounds into the individual wells. If a compound has no effect on PDE activity or on any component of the yeast cAMP pathway, the cells of both strains deplete their cAMP leading to increased fbp1-ura4 transcription, which inhibits growth in the presence of 5FOA. If a compound stimulates cAMP production by targeting a component of the yeast cAMP pathway or inhibits fbp1-ura4 expression in a cAMP-independent manner, both strains display enhanced 5FOA-resistant growth to a similar degree. If a compound is an inhibitor of the exogenous PDE, the cAMP levels rise in the experimental strain, but not in the control strain, leading to differential 5FOA-resistant growth. Growth of the experimental and control strains are measured by measuring optical density. The effect of a compound is independently verified by measuring β-galactosidase expression from the fbp1-lacZ reporter in the experimental strain and by direct measurement of cAMP levels.

Example 4 A Fission Yeast-Based High Throughput Screen to Identify Chemical Modulators of cAMP Phosphodiesterase

Described herein is a fission yeast-based platform to detect compounds that either inhibit or activate heterologously-expressed cAMP phosphodiesterases (PDEs) that is suitable for high throughput drug screening. PDEs comprise a superfamily of enzymes that serve as drug targets in a variety of human diseases. The utility of this system is demonstrated by the construction and characterization of strains that express mammalian PDE2A, PDE4A, PDE4B, and PDE8A and respond appropriately to treatment with known PDE2A and PDE4 inhibitors. High throughput drug screens of two bioactive compound libraries were successfully conducted for PDE inhibitors using strains expressing PDE2A, PDE4A, PDE4B, and the yeast PDE Cgs2, demonstrating the ability of this system to determine PDE specificity through parallel screens of strains expressing distinct enzymes. The use of this platform to identify both chemical activators of PDEs, as well as genes that encode biological modulators of PDEs, which could serve as targets for future drug screens, is also discussed.

INTRODUCTION

Cyclic AMP (cAMP) signaling pathways are employed by unicellular organisms and metazoan cells to transduce signals from a cell's surroundings to elicit appropriate responses. Unicellular organisms generally use this pathway to control metabolism and sexual development, often as a function of carbon source signaling. Mammalian cells produce cAMP signals in response to the detection of a variety of molecules including hormones, odorants, and neurotransmitters. This signaling pathway in mammals is complicated due to the presence of multiple cAMP-producing adenylyl cyclases and cAMP-destroying cAMP phosphodiesterases (PDEs)^(1, 2).

There are 11 families of mammalian PDEs encoded by 21 genes, which produce more than 100 isoenzymes^(2, 3). PDEs from the PDE4, PDE7, and PDE8 families specifically act on cAMP, PDEs from the PDE1, PDE2, PDE3, PDE10, and PDE11 families act on both cAMP and cGMP, while PDEs from the PDE5, PDE6, and PDE9 families act preferentially on cGMP. The presence of multiple PDE isoenzymes in various tissues complicates efforts to determine the relative roles of specific enzymes in any given biological process. Even so, chemical inhibitors of PDEs, and in some cases chemical activators, are seen as potential therapeutic compounds for the treatment of a variety of conditions including anxiety, depression, Alzheimer's disease, Parkinson's disease, Huntington's disease, schizophrenia, psychosis, sepsis, asthma, chronic obstructive pulmonary disease, pulmonary hypertension, renal disease, stroke, rhinitis, psoriasis, memory loss, chronic lymphocytic leukemia, prostate cancer, thyroid disease, male hypogonadism, cardiac disease, diabetes, obesity, multiple sclerosis, rheumatoid arthritis, penile erectile dysfunction, osteoporosis and cystic fibrosis²⁻⁹. Described here is an in vivo screen for identifying both chemical inhibitors and activators of cAMP PDEs using a simple growth assay in the fission yeast Schizosaccharomyces pombe.

Previous studies on S. pombe glucose/cAMP signaling made use of two reporters whose expression is driven by the glucose-repressible fbp1⁺ promoter¹⁰. The fbp1-ura4 reporter places uracil biosynthesis under the control of the glucose/cAMP pathway, such that cells with high cAMP levels from glucose signaling cannot grow in medium lacking uracil (SC-ura), but do grow in medium containing the pyrimidine-analog 5-fluoro-orotic acid (5FOA), due to repression of the reporter (FIG. 3A). In contrast, cells with low cAMP levels from defects in glucose signaling grow in medium lacking uracil, but die in 5FOA medium, due to expression of the reporter (FIG. 3B). The second reporter, fbp1-lacZ, allows for easy quantitation of expression from the fbp1⁺ promoter. It is shown herein that strains expressing the mammalian enzymes PDE2A, PDE4A, PDE4B, and PDE8A produced functional PDEs whose activities affected the expression of these fbp1-driven reporters. In addition, reporter expression in PDE4A- and PDE4B-expressing strains was repressed by the PDE4 inhibitor rolipram, while reporter expression in a PDE2A-expressing strain was repressed by the PDE2A inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). Successful high throughput drug screens for chemical inhibitors of the PDE2A, PDE4A, PDE4B, and yeast PDE Cgs2 have validated the utility of this platform. Also described, are additional capabilities of this screening platform to identify chemical activators of PDEs, as well as genes that encode biological activators or inhibitors of PDEs, which can serve as target proteins in future drug screens. The flexibility and versatility of this system demonstrate that the screen is an effective way to identify both chemical and biological modulators of PDEs from a variety of organisms.

Methods

Some yeast strains used are listed in Table 3. For the values in Table 3 β-galactosidase activity was determined from two to three independent exponential phase cultures. The average ±SD represents specific activity per milligram of soluble protein.

TABLE 3 β-galactosidase activity from fbp1-lacZ expression in gpa2⁻ mutant strains Strain PDE β-galactosidase activity CHP861 Cgs2⁺ 2537 ± 292 LWP364 PDE2A 331 ± 28 CHP1098 PDE4A 1383 ± 269 DIP72 PDE4B 825 ± 70 DDP13 PDE8A  473 ± 139 LWP98 Cgs2-2 40 ± 4

Methods for the growth and transformation of fission yeast have been previously described¹⁹. The murine PDE genes were amplified by PCR using oligonucleotides containing approximately 60 nt of sequence flanking the S. pombe cgs2⁺ gene to direct homologous recombination to this locus. The recipient strain carries a ura4⁺-marked disruption of cgs2⁺²⁹ (also referred to as pde1⁺) to allow for 5FOA-counterselection for candidate transformants. PCR was used to confirm the homologous integration events. Subsequent strains were constructed by standard genetic crosses and tetrad dissection to introduce the fbp1-lacZ and fbp1-ura4 reporters, as well as the pap1Δ allele.

β-galactosidase assays and characterization of 5FOA-sensitivity were carried out as previously described¹⁰. cAMP assays were performed on exponential phase cells grown in EMM complete medium (3% glucose), using the Assay Designs cAMP EIA kit, according to manufacturer's instructions (Assay Designs, Ann arbor, Mich.).

High throughput drug screens were carried out at the Broad Institute's Chemical Biology Program screening facility (Broad Institute, Cambridge, Mass.). Depending upon the strain, cultures were pregrown to exponential phase in EMM complete medium containing from 0.5 to 2.5 mM cAMP to repress fbp1-ura4 transcription. Cells were collected by centrifugation, resuspended in 5FOA medium, and 25 μl were transferred to 384-well microtiter dishes (untreated, with flat clear bottoms) that had been pre-filled with 25 μl 5FOA medium and pre-pinned with 100 nl of compounds (stock solutions were generally 10 mM) from a subset of the Prestwick Bioactive and the Microsource Spectrum compound libraries. Starting cell concentrations ranged from 0.5×10⁵ to 4×10⁵ cells/ml depending on the screening strain. As appropriate, control plates received either 100 nl 10 mM rolipram or DMSO. Other positive control dishes contained 5 mM cAMP in the 5FOA medium. Cultures were grown for 48 hours at 30° C., sealed in an airtight container with moist paper towels to prevent evaporation. Optical densities (OD₆₀₀) of cultures were measured using a microplate reader. Bioinformatic analysis of the results to determine composite Z scores was performed as previously described^(30, 31).

Results

To develop yeast strains whose growth behaviors could serve as a reflection of the activity of heterologously-expressed PDEs, homologous recombination was used to replace the only S. pombe PDE gene, cgs2⁺, with each of four murine PDE genes, PDE2A, PDE4A, PDE4B, and PDE8A¹¹⁻¹³. Strains expressing these enzymes do not display the severe mating defect associated with the loss of PDE activity¹⁴, indicating that these PDEs are functional when expressed in S. pombe.

Next, strains were constructed that expressed the murine PDEs together with the fbp1-driven reporters, and carried mutant alleles of either the git3⁺ glucose receptor gene or the gpa2⁺ Gα subunit gene, both of which were required for glucose detection, adenylyl cyclase activation, and transcriptional repression of the fbp1-ura4 and fbp1-lacZ reporters¹⁵⁻¹⁸. The relative level of reporter expression in these strains reflected the activity of the PDEs expressed. β-galactosidase activity in the gpa2⁻ mutant strains, as compared with similar strains expressing either the wild-type S. pombe Cgs2⁺ PDE or the frame-shifted, and presumably inactive, Cgs2-2 truncated PDE¹⁹ demonstrated that all four murine PDEs were active in S. pombe (Table 1). The relative level of PDE activity, as reflected by the degree to which β-galactosidase activity was elevated by the reduction in cAMP levels, was Cgs2⁺>PDE4A>PDE4B>PDE8A≈PDE2A>Cgs2-2. This order of activity was consistent with the ability of git3⁻ and gpa2⁻ mutations to confer 5FOA-sensitive (5FOA^(S)) growth to strains expressing the murine PDEs (see below).

The effect of known PDE inhibitors on the expression of the fbp1-lacZ fusion in murine PDE-expressing strains was tested. As seen in Table 4, rolipram, a PDE4 inhibitor, reduced β-galactosidase activity in PDE4A- and PDE4B-expressing cells, but not in Cgs2- or PDE8A-expressing cells. These results supported previous studies indicating that PDE8A was insensitive to rolipram. In addition, the PDE2A inhibitor EHNA reduced β-galactosidase activity expressed from a PDE2A strain (Table 4). For Table 4 β-galactosidase activity was determined from 3 to 4 independent exponential phase cultures. The average±SD represents specific activity per milligram of soluble protein. PDE8A was not able to be inhibited with dipyridamole, which has been shown to inhibit PDE8A¹², and this result may have been due to a permeability problem in the yeast.

TABLE 4 β-galactosidase activity in response to PDE inhibitor treatment β-galactosidase activity 50 μM 100 μM Strain PDE Vehicle Rolipram Rolipram CHP861 Cgs2 1661 ± 121  1807 ± 446 1784 ± 429 DDP26 PDE4A 998 ± 154 271 ± 30 162 ± 17 DIP72 PDE4B 432 ± 170  32 ± 12 21 ± 7 DDP13 PDE8A 241 ± 61  253 ± 46 237 ± 67 LWP98 Cgs2-2 23 ± 10 19 ± 9  20 ± 11 β-galactosidase activity 5 μM 20 μM 200 μM Strain PDE Vehicle EHNA EHNA EHNA LWP367 PDE2A 587 ± 7 473 ± 19 197 ± 51 45 ± 3 In an effort to increase the sensitivity to PDE inhibitors, further experiments included examination of whether deleting pap1⁺, encoding a zinc finger transcriptional activator required for ABC transporter expression and whose overexpression confers staurosporine-resistance^(20, 21), enhanced inhibition of PDE4A by rolipram. As shown in FIG. 4, PDE4A-expressing cells lacking pap1⁺ (pap1Δ), were more sensitive to rolipram than pap1⁺ cells. Moreover, pap1Δ strains that were 5FOA^(S) due to low cAMP levels maintained the 5FOA^(S) growth phenotype for longer periods of incubation than equivalent pap1⁺ strains. Such enhanced sensitivity to 5FOA is useful to help in the detection of compounds that confer 5FOA^(R) growth due to PDE inhibition.

To determine if the effect of rolipram on PDE4-expressing cells and of EHNA on PDE2A-expressing cells was through inhibition of the heterologously-expressed PDEs, cAMP levels were measured before and after drug treatment. As shown in FIG. 5A, cAMP levels increased within 10 minutes of exposure to 200 μM inhibitor and reached peak levels within one hour. Additional experiments were performed to examine whether varying degrees of PDE inhibition could be detected by measuring cAMP levels at the one-hour time point in cells exposed to lower concentrations of inhibitor. FIG. 5B shows that PDE4A was only partially inhibited by 20 μM rolipram, while PDE4B was completely inhibited at this concentration, suggesting that PDE4B was more sensitive than PDE4A to rolipram in this system. Furthermore, cAMP levels in a strain expressing PDE8A were completely insensitive to rolipram treatment, consistent with previous studies of PDE8A¹², and also indicating that rolipram does not affect cAMP generation in fission yeast. Finally, PDE2A showed partial inhibition by EHNA at 20 μM as compared to 200 μM EHNA. Thus, PDE inhibition can be indirectly quantitated by measuring the effect of a compound on cAMP levels in target yeast strains.

Although the fbp1-lacZ reporter allowed for a measurement of PDE inhibition, the true power of this system is in the growth phenotype conferred by transcription of the fbp1-ura4 reporter. PDE inhibitors should restore 5FOA^(R) growth to strains possessing low basal cAMP levels by elevating cAMP levels to repress fbp1-ura4 transcription (FIG. 3D). Conversely, PDE activators should confer growth in SC-ura medium to strains possessing high cAMP levels by reducing cAMP levels to increase fbp1-ura4 transcription (FIG. 3C). As mentioned above, mutations in either the git3⁺ or gpa2⁺ genes were introduced into various PDE-expressing strains. While a gpa2⁻ mutant allele conferred 5FOA-sensitivity on PDE2A-, PDE4A-, PDE4B-, and PDE8A-expressing strains, only Cgs2- and PDE4A-expressing strains became 5FOA^(S) when carrying a mutant allele of git3⁺. These results are consistent with previous observations that loss of Gpa2 confers a greater defect in cAMP signaling than does loss of Git3^(10, 17, 18), and that Cgs2 and PDE4A were more active than the other three PDEs in the strains used (Tables 3 and 4).

To determine whether the 5FOA growth phenotype could be exploited for high throughput drug screening, strains expressing PDE2A, PDE4A, PDE4B, or PDE8A were pre-grown in EMM medium containing cAMP and then transferred to 5FOA medium in 384 well microtiter plates in the presence or absence of cAMP. OD₆₀₀ measurements were taken after 48 hours incubation at 30° C. In each strain, the addition of cAMP to the growth medium restored 5FOA^(R) growth. Similar experiments in which 20 μM rolipram (final concentration) was pinned into 192 of the 384 wells, in place of cAMP addition to the medium, produced 5FOA^(R) growth in the PDE4A and PDE4B-expressing strains. For example, in a typical experiment with CHP1113 cells (PDE4B), the OD₆₀₀ of the rolipram-treated cultures was 1.28+/−0.07 while the OD₆₀₀ of the untreated wells was 0.18+/−0.02. When using CHP1098 cells (PDE4A), the OD₆₀₀ of the rolipram-treated cultures was 1.15+/−0.06, while the OD₆₀₀ of the untreated wells was 0.2+/−0.03. The Z factors (a statistical assessment of the quality of datasets used in high throughput screening²²) for these screens are 0.76 and 0.72, respectively, placing them well above the 0.5 minimum Z factor indicative of a robust screen.

As a final test of the utility of this system, screening was performed on a pair of libraries containing 3,120 bioactive compounds, including known PDE inhibitors, using 5FOA^(S) strains expressing PDE2A, PDE4A, PDE4B, or Cgs2 for compounds that confer 5FOA^(R) growth. Duplicate plates were screened and compounds that confer 5FOA^(R) growth with composite Z scores of ≧8.53 (the cut-off used by the Broad Institute's Chemical Biology Program, where the screens were performed) were identified.

Discussion

This Example describes a novel fission yeast cell-based screening platform, amenable for high throughput drug screening to identify compounds that alter PDE activity. While a budding yeast system based on heat shock sensitivity of stationary phase cells has been previously reported²³, cells in that assay had to be exposed to 0.5 mM to 2 mM rolipram to detect an effect on PDE4B and was not amenable to a high throughput screening format^(24, 25). In contrast, using these new assay methods has permitted successful screening of compound libraries at an average concentration of 20 μM to detect both known and previously unidentified PDE inhibitors. This is a relatively inexpensive assay, and permits development of a large collection of strains expressing either mammalian cAMP-specific or dual-specificity PDEs. This platform is also used with PDEs from pathogens, whose inhibition may either kill the target pathogen or reduce virulence. Strains expressing a broad panel of PDEs are used to identify compounds possessing desirable specificity profiles to suggest the potential of individual compounds as candidate therapeutics. Moreover, because this platform identifies compounds based on stimulation of cell growth, it will not detect compounds that, while inhibiting PDEs in vitro, are too cytotoxic or cell-impermeable for therapeutic use. This is not the case for the majority of PDE assays, which are carried out in vitro on purified proteins or on protein extracts. In addition, this in vivo screening platform should be able to detect PDE inhibitors that may not be identified by in vitro screens. For example, compounds that prevent either intermolecular or intramolecular interactions required for enzyme formation would be overlooked in an in vitro assay on purified enzymes or protein extracts, yet should be identifiable in this assay.

High throughput screens against 3,120 bioactive compounds using strains expressing the yeast PDE Cgs2, or the murine PDEs 2A, 4A, and 4B identified a number of compounds that promote 5FOA^(R) growth, presumably by inhibiting the target PDEs to raise cAMP levels. These included the known PDE4 inhibitors rolipram and zardaverine, which only affected the PDE4A- and PDE4B-expressing strains. Other compounds identified in the screens are members of the coumarin, furocoumarin, and flavonoid families that are known to have PDE inhibitory properties (see review by Peluso, 2006²⁶). For example, the screens identified the furocoumarins trioxsalen, khellin, and visnagin, which are known PDE inhibitors^(27, 28). In addition, the relative overlap of the compounds identified in each screen further validated this platform.

The ability to identify PDE inhibitors is based on the growth phenotype conferred by the cAMP-repressible fbp1-ura4 reporter. This system can also identify compounds that stimulate PDE activity to lower cAMP levels and increase fbp1-ura4 expression. PDE activators should confer Ura⁺ growth to strains whose high basal cAMP levels repress fbp1-ura4 expression in the absence of drug exposure (FIG. 3C). Finally, as yeast are capable of maintaining autonomously-replicating plasmids, one can screen cDNA libraries for genes that encode biological inhibitors or activators of target PDEs, which can serve as novel targets for high throughput drug screens. Thus, this screening platform can be used to identify novel PDE inhibitors and activators, as well as new ways to moderate cAMP signaling pathways in an effort to improve therapeutic approaches to treating a wide array of human diseases.

REFERENCES FOR EXAMPLE 4

-   1. Kamenetsky, M. et al. J Mol Biol 362, 623-639 (2006). -   2. Bender, A. T. & Beavo, J. A. Pharmacol Rev 58, 488-520 (2006). -   3. Lerner, A. & Epstein, P. M. Biochem J 393, 21-41 (2006). -   4. Vasta, V., et al., Proc Natl Acad Sci USA 103; 19925-19930     (2006). -   5. Dyke, H. J. & Montana, J. G. Expert Opin Investig Drugs 11, 1-13     (2002). -   6. Boswell-Smith, et al., Br J Pharmacol 147 Suppl 1, S252-257     (2006). -   7. O'Donnell, J. M. & Zhang, H. T. Trends Pharmacol Sci 25, 158-163     (2004). -   8. Lugnier, C. Pharmacol Ther 109, 366-398 (2006). -   9. Hebb, A. L. & Robertson, H. A. Curr Opin Pharmacol (2006). -   10. Hoffman, C. S. & Winston, F. Genetics 124, 807-816 (1990). -   11. Cherry, J. A., et al., Biochim Biophys Acta 1518, 27-35 (2001). -   12. Soderling, S. H., et al., Proc Natl Acad Sci USA 95, 8991-8996     (1998). -   13. Wu, A. Y., et al. J Biol Chem 279, 37928-37938 (2004). -   14. DeVoti, J., et al., Embo J 10, 3759-3768 (1991). -   15. Hoffman, C. S. Biochem Soc Trans 33, 257-260 (2005). -   16. Ivey, F. D. & Hoffman, C. S. Proc Natl Acad Sci USA 102,     6108-6113 (2005). -   17. Nocero, M., et al. Genetics 138, 39-45 (1994). -   18. Welton, R. M. & Hoffman, C. S. Genetics 156, 513-521 (2000). -   19. Wang, L., et al., Genetics 171, 1523-1533 (2005). -   20. Toone, W. M. et al. Genes Dev 12, 1453-1463 (1998). -   21. Toda, T., et al., Genes Dev 5, 60-73 (1991). -   22. Zhang, J. H., et al., J Biomol Screen 4, 67-73 (1999). -   23. Colicelli, J. et al. Proc Natl Acad Sci USA 88, 2913-2917     (1991). -   24. Pillai, R., et al., Proc Natl Acad Sci USA 90, 11970-11974     (1993). -   25. Atienza, J. M. & Colicelli, J. Methods 14, 35-42 (1998). -   26. Peluso, M. R. Exp Biol Med (Maywood) 231, 1287-1299 (2006). -   27. Duarte, J. et al. Gen Pharmacol 32, 71-74 (1999). -   28. Bovalini, L. et al. Z Naturforsch [C] 42, 1009-1010 (1987). -   29. Mochizuki, N. & Yamamoto, M. Mol Gen Genet 233, 17-24 (1992). -   30. Kim, Y. K. et al. J Am Chem Soc 126, 14740-14745 (2004). -   31. Franz, A. K., et al., J Am Chem Soc 129, 1020-1021 (2007).

Example 5 Methods for Preparing Yeast Strains Containing Exogenous PDEs

These methods can be used to prepare fission yeast strains that lack endogenous cAMP PDEs and that include one or more exogenous PDE. Conditions to promote growth and to optimize cAMP levels for any specific strain generated may be determined using methods in the art and/or methods described herein.

This example provides protocols that have been and can be used to introduce PDE genes into the fission yeast. The resulting yeast strains are useful in screening methods and assays for cAMP PDE activators and inhibitors.

PDE genes were introduced into the fission yeast PDE gene locus (cgs2⁺) by PCR amplification of the gene to be introduced using oligonucleotides that contain sequences that flank the cgs2 gene. The PCR product was used to transform strain JZ666, which contains a ura4⁺-marked deletion of cgs2, which allowed for 5FOA-counterselection to identify colonies that have lost the ura4 gene due to its replacement by the PDE gene through homologous recombination. The host strain is homothallic (cells from the same strain are capable of mating with each other), however mating of this strain is defective due to the high cAMP levels conferred by the disruption of the cgs2 PDE gene. An initial screen for candidates that received a foreign PDE gene was carried out by either microscopic examination of cells growing on defined medium (Edinburgh minimal medium (EMM) for example) or by exposing plates to iodine vapors, which stain asci that are produced by mating. A second feature of reducing cAMP levels is that cells show improved survival in stationary phase. This was and can be screened for by microscopy or by replica plating colonies from plates that have been incubated for as much as one week to a fresh plate, and by examining the efficiency with which cells from individual colonies are able to grow and form new colonies. Candidate colonies from either method are further examined by PCR to detect the homologous recombination event that would introduce the foreign PDE gene into the cgs2⁺ locus.

Because homologous recombination is not as efficient in S. pombe as it is in budding yeast, an alternative strategy has also been employed to introduce PDE genes into the cgs2 locus. Rather than directly introducing the PCR product into the chromosomal locus, JZ666 cells were co-transformed with the PCR product and a linearized plasmid that carries the ura4-marked disruption of cgs2. By digesting the plasmid within the ura4 gene, homologous recombination between the plasmid and the PCR product was stimulated. The PDE gene recombines into the plasmid through the process of gap repair at a higher efficiency than seen for recombination into the chromosome. Cells carrying plasmids that express the PDE were identified as described above. Once the plasmid had been rescued to E. coli and a plasmid preparation was obtained, the plasmid was digested with one or two restriction enzymes to produce a fragment containing the PDE gene along with 500 to 2000 base pairs of cgs2 flanking sequences. This fragment was used to introduce the PDE gene into the cgs2 chromosomal locus in strain JZ666 by homologous recombination. This was more efficient than the direct transformation with a PCR product (described above) because this fragment possesses significantly more targeting sequences at its ends.

For the design of oligonucleotides for PCR, the 5′ end of each oligonucleotide should contain approximately 60 nucleotides from the following sequences that flank cgs2.

Forward targeting sequence (the final ATG repre- sents the Cgs2 START codon) (SEQ ID NO: 1) 5′TCTCCACATTTCGAGCATCGTTTATCGTACCCTAAATCTACGGTAGTA AATGTATGCTTGTAATAAATATGACGTCAACCGACATGTTTTTGTAGACT AGTGCATGCACCGGAGATCTGTAACTCTCCATAAGCCTAGCCATG 3′ Reverse targeting sequence (SEQ ID NO2) 5′AAGCGAGGTACGATGAACTGGTAATGAAAAATAAAAAAAGGTAATAAT TAATGCTTTAGCATTCAATAATTAACAACAAAGTCAAAATTCCTCCAACA G 3′ As a specific example, to introduce the human PDE4D3 gene into the cgs2 locus, the following two oligonucleotides were used to PCR amplify PDE4D3 from a plasmid carrying this cDNA.

Forward oligonucleotide (SEQ ID NO: 3) TGTTTTTGTAGACTAGTGCATGCACCGGAGATCTGTAACTCTCCATAAGC CTAGCCATGATGCACGTGAATAATTTTCCC Reverse oligonucleotide (SEQ ID NO: 4) TAATAATTAATTGCTTTAGCATTCAATAATTAACAACAAAGTCAAAATTC CTCCAACAGTTACGTGTCAGGAGAACGATC This approach has been successfully used with the following PDE genes and is used with additional PDE genes. Murine PDE1C4 (Genbank Accession number L76947) Murine PDE2A (Genbank Accession number NM_(—)001008548) Murine PDE3B (Genbank Accession number AF547435) Murine PDE4A1 (Genbank Accession number NM_(—)019798) Rat PDE4A5 (Genbank Accession number L27057) Murine PDE4B3 (Genbank Accession number NM_(—)019840) Human PDE4D3 (Genbank Accession number U50159) Human PDE7A (Genbank Accession number L12052) Murine PDE8A (Genbank Accession number BC132145) Trypanosoma brucei PDEB1 (Genbank Accession number AY028446) Trypanosoma brucei PDEB2 (Genbank Accession number XM_(—)798722) Trypanosoma cruzi PDEB1 (Genbank Accession number AY099403) Human PDE10A (Genbank Accession number NM_(—)006661) The sequences of oligonucleotide primers used in the construction of the strains are provided in Table 5.

TABLE 5 PDE gene Accession PDE1C4 L76947 Forward CATGTTTTTGTAGACTAGTGCATGCACCGGAGATCTGTAAC TCTCCATAAGCCTAGCCATGGAGTCTCCAACCAAGGAAA (SEQ ID NO: 5) Reverse AATGAAAAATAAAAAAAGGTAATAATTAATTGCTTTAGCAT TCAATAATTAACAACAAAGTCAAAATTCCTCCAACAGTTAT CCGTAGTCTCCTGGCAAG (SEQ ID NO: 6) PDE2A NM_001008548 Forward ACATGTTTTTGTAGACTAGTGCATGCACCGGAGATCTGTAA CTCTCCATAAGCCTAGCCATGGGGCAGGCATGCGGCCAC (SEQ ID NO: 7) Reverse ATAATTAATTGCTTTAGCATTCAATAATTAACAACAAAGTC AAAATTCCTCCAACAGTCAGCCCTCGAGGCTGCAGCAGC (SEQ ID NO: 8) PDE3B AF547435 Forward ACATGTTTTTGTAGACTAGTGCATGCACCGGAGATCTGTAA CTCTCCATAAGCCTAGCCATGAGGAAAGACGAGCGCGAG (SEQ ID NO: 9) Reverse* TAATAATTAATTGCTTTAGCATTCAATAATTAACAACAAAG TCAAAATTCCTCCAACAGAGGCCTGAATTCCTCGAGGTC (SEQ ID NO: 10) PDE4A1 NM_019798 Forward ACATGTTTTTGTAGACTAGTGCATGCACCGGAGATCTGTAA CTCTCCATAAGCCTAGCCATGCCTCTGGTTGACTTCTTC (SEQ ID NO: 11) Reverse AAATTAAAAAAAAAAAATAAAAATATAATGAATATATGACC ATGACCCTGGGATGCTATTAGGCAGGGTCTCCACCTGAC (SEQ ID NO: 12) PDE4A5 L27057 Forward ATGTTTTTGTAGACTAGTGCATGCACCGGAGATCTGTAACT CTCCATAAGCCTAGCCATGAGCCATGGAGCCTCCGGCCG (SEQ ID NO: 13) Reverse AATAATTAATTGCTTTAGCATTCAATAATTAACAACAAAGT CAAAATTCCTCCAACAGTCAGGCAGGGTCTCCGCCTGAC (SEQ ID NO: 14) PDE4B3 NM_019840 Forward GACATGTTTTTGTAGACTAGTGCATGCACCGGAGATCTGTA ACTCTCCATAAGCCTAGCCATGACAGCAAAAAATTCTCC (SEQ ID NO: 15) Reverse ATTAAAAAAAAAAAATAAAAATATAATGAATATATGACCAT GACCCTGGGATGCTACTAAACTCTAGATATTCAACAGGC (SEQ ID NO: 16) PDE4D3 U50159 Forward TGTTTTTGTAGACTAGTGCATGCACCGGAGATCTGTAACTC TCCATAAGCCTAGCCATGATGCACGTGAATAATTTTCCC (SEQ ID NO: 3) Reverse TAATAATTAATTGCTTTAGCATTCAATAATTAACAACAAAG TCAAAATTCCTCCAACAGTTACGTGTCAGGAGAACGATC (SEQ ID NO: 4) PDE7A L12052 Forward* ACATGTTTTTGTAGACTAGTGCATGCACCGGAGATCTGTAA CTCTCCATAAGCCTAGCCGGACGGCCTCCGAAACCATG (SEQ ID NO: 17) Reverse AAAAAGGTAATAATTAATTGCTTTAGCATTCAATAATTAAC AACAAAGTCAAAACCTTATGATAACCGATTTTCCTGAGG (SEQ ID NO: 18) PDE8A BC132145 Forward AAATATGACGTCAACCGACATGTTTTTGTAGACTAGTGCAT GCACCGGAGATCTGTAACTCTCCATAAGCCTAGATGGGC (SEQ ID NO: 19) Reverse GGTAATAATTAATTGCTTTAGCATTCAATAATTAACAACAA AGTCAAAATTCCTCCAACAGGCAGCTCTGGCTAACAGTG (SEQ ID NO: 20) T. brucei PDEB1 AY028446 Forward ATGTTTTTGTAGACTAGTGCATGCACCGGAGATCTGTAACT CTCCATAAGCCTAGCCATGTTCATGAACAAGCCCTTTGG (SEQ ID NO: 21) Reverse* AGGTAATAATTAATTGCTTTAGCATTCAATAATTAACAACA AAGTCAAAATTCCTCCAACAGTCGAGGCTGATCAGCGGG (SEQ ID NO: 22) T. brucei PDEB2 XM_798722 Forward CATGTTTTTGTAGACTAGTGCATGCACCGGAGATCTGTAAC TCTCCATAAGCCTAGCCATGACACACAACGGTGGTCGTC (SEQ ID NO: 23) Reverse AGGTAATAATTAATTGCTTTAGCATTCAATAATTAACAACA AAGTCAAAATTCCTCCAACAGTCGAGGCTGATCAGCGGG (SEQ ID NO: 24) T. cruzi PDEB1 AY099403 Forward ACATGTTTTTGTAGACTAGTGCATGCACCGGAGATCTGTAA CTCTCCATAAGCCTAGCCATGGGGCAGGCATGCGGCCAC (SEQ ID NO: 25) Reverse AGGTAATAATTAATTGCTTTAGCATTCAATAATTAACAACA AAGTCAAAATTCCTCCAACAGTCGAGGCTGATCAGCGGG (SEQ ID NO: 26) Homo sapiens PDE10A NM_006661 Forward* GACATGTTTTTGTAGACTAGTGCATGCACCGGAGATCTGTA ACTCTCCATAAGCCTAGCCGGCACCAAAATCAACGGGAC (SEQ ID NO: 27) Reverse* GTAATAATTAATTGCTTTAGCATTCAATAATTAACAACAAA GTCAAAATTCCTCCAACAGTTATT AGGACAAGGCTGGTG (SEQ ID NO: 28) *Oligonucleotide is designed to prime off of the vector sequence rather than the sequence of the PDE gene.

Once the PDE gene was introduced into the cgs2 locus, drug screening strains were constructed by standard genetic crosses with strains that contain the following genetic features.

1. fbp1-ura4 fusion: This is the reporter that produces the cAMP-dependent growth characteristics. 2. fbp1-lacZ fusion: While not necessary for high throughput screening, this reporter allows easy quantitation of expression from the fbp1 promoter, which can be useful for characterizing the effect of adding candidate compounds or cAMP or cGMP to the growth medium (see below). 3. pap1Δ: The deletion of the pap1⁺ gene is not essential for high throughput screening, however it appears to make the cells more sensitive to both 5FOA and to drug treatment. This gene encodes a transcriptional activator that regulates the expression of ABC transporter genes. Loss of this gene may allow compounds to accumulate in S. pombe. 4. A mutation in a glucose/cAMP pathway gene: This was required for most, but not all strains in order to screen for PDE inhibitors. Mutations such as git3-14 and git11Δ cause a modest reduction in cAMP generation, which the git3Δ deletion causes a moderate reduction in cAMP generation, and the gpa2 disruption causes a significant reduction in cAMP generation. In order to carry out a PDE inhibitor screen, cells must be 5FOA-sensitive due to an insufficient cAMP level to repress fbp1 transcription. These various mutations were used to control cAMP levels.

Should a PDE be encountered that has such low activity that even loss of the gpa2 gene fails to confer 5FOA-sensitivity, there are two alternative strategies to develop a screening strain. One strategy includes introducing the PDE gene into S. pombe under the control of a stronger promoter than the cgs2 promoter. Such promoters can be the nmt1, nmt41 or the SV40 promoter. A second strategy includes introducing a deletion of the adenylate cyclase git2 gene into the strain so that there is no cAMP production. Such cells are 5FOA-sensitive regardless of the strength of the heterologously-expressed PDE gene (as shown FIG. 1, which indicates that a git2Δ cgs2-s1 mutant is 5FOA-sensitive). In this case, one can determine a concentration of cAMP that is added to the medium to confer 5FOA-resistant growth to a strain lacking both adenylate cyclase and PDE activity, but is insufficient to confer growth to a strain that lacks adenylate cyclase, but expresses the weak target PDE. A PDE inhibitor is identified by its ability to re-establish 5FOA-resistant growth due to the addition of this low level of cAMP. To summarize, if a PDE is extremely weak, one can replace endogenous cAMP production with exogenous cAMP addition to give one complete control over the level of cAMP in the system.

Table 6 describes growth conditions prior to exposure to 5FOA medium that have been determined for various strains. Optimized growth conditions for additional strains can be determined using routine culture methods.

TABLE 6 Experimental Conditions Per PDE Strain Pregrowth (mM Cell Density Strain PDE cAMP + EMM) (cell/ml) CHP1113 PDE4B3 0.5 5 × 10{circumflex over ( )}4 CHP932 Cgs2 2.5 1 × 10{circumflex over ( )}5 LWP369 PDE2A 0.2 5 × 10{circumflex over ( )}4 CHP1098 PDE4A1 1 4 × 10{circumflex over ( )}5 CHP1155 PDE4A5 2.5 2 × 10{circumflex over ( )}5 CHP1169 PDE7A 2.5 1 × 10{circumflex over ( )}5 DDP16 PDE8A 0.5 2 × 10{circumflex over ( )}4 CHP1167 PDE4D3 0.5 mM TBD cGMP* CHP1179 PDE1C4 1.1 mM TBD cGMP* *Situations in which exogenous cAMP is not able to confer 5FOA-resistant growth have been observed, however cGMP can be used successfully. TBD—To be determined.

Method for PDE Inhibitor Screen

The following provides a general protocol for PDE inhibitor screening. Such a method, or similar methods are useful to screen the strains of the invention to identity PDE inhibitors.

Cells were pregrown in EMM medium [MP Biomedicals (Solon, Ohio), 3% glucose, filter-sterilized to avoid carmelization, which would introduce variability into the optical density of the medium] containing from 0 mM to 2.5 mM cAMP (or either 0.5 mM or 1.0 mM cGMP). This was to repress expression of the fbp1-ura4 reporter prior to exposure of cells to 5FOA medium. Cells were grown at 30° C. to exponential phase (approximately 10⁷ cells/ml). Cells were collected by centrifugation and resuspended in 5FOA medium, and 25 μl were transferred to 384-well microtiter dishes (untreated, with flat clear bottoms) that had been pre-filled with 25 μl 5FOA medium and pre-pinned with 100 nl of compounds (stock solutions were generally 10 mM). Starting cell concentrations ranged from 0.5×10⁵ to 4×10⁵ cells/ml depending on the screening strain. As appropriate, control plates received either 100 nl 10 mM rolipram (for rolipram-sensitive PDE4s) or DMSO. Other positive control dishes contained 5 mM cAMP in the 5FOA medium for PDEs that lack appropriate control compounds. Cultures were grown for 48 hours at 30° C., sealed in an airtight container with moist paper towels to prevent evaporation. Optical densities (OD₆₀₀) of cultures were measured using a microplate reader. Bioinformatic analysis of the results to determine composite Z scores was performed as previously described (1, 3).

REFERENCES FOR EXAMPLE 5

-   1. Franz, A. K., et al., J Am Chem Soc 129:1020-1. -   2. Hoffman, C. S., and R. Welton. 2000. Biotechniques 28:532-6, 538,     540. -   3. Kim, Y. K., et al., J Am Chem Soc 126:14740-5.

Example 6 Methods of Expressing a cAMP PDE at a Higher Level than from the Yeast PDE Promoter

The method includes the introduction of a PDE into the plasmid pRH1 (Hoffman and Hoffman 2006), which carries two selectable markers. It has the S. cerevisiae LEU2 gene that complements S. pombe leu1 mutations and is transcribed from the SV40 promoter. It also has the S. pombe lys2 gene. The PDE gene is introduced into pRH1, replacing the LEU2 gene by gap repair transformation (Wang, Kao et al. 2004), so that the PDE gene is expressed from the SV40 promoter (this gives high level expression). Specifically, this is done by linearizing pRH1 within the LEU2 gene with an enzyme such as BbsI that cuts in LEU2, but not elsewhere in the plasmid. This linearized plasmid is co-transformed into a lys2⁻ mutant strain of S. pombe together with a PCR product that contains the PDE gene flanked by sequences from pRH1 that target the PDE gene to recombine with the plasmid upon uptake into the yeast cells. For example, to integrate clones obtained from the company OriGene, using priming sequences that are universal to the cloning vector, the following oligonucleotides are used:

Forward oligonucleotide (SEQ ID NO: 29) 5′ ttccagaagtagtgaggaggcttttttggaggcctaggcttttgcaa aaagctttgcaaaggcaccaaaatcaacgggac 3′ Reverse oligonucleotide (SEQ ID NO: 30) 5′ tgaatgggcttccatagtttgaaagaaaaaccctagcagtactggca agggagacattccttattaggacaaggctggtg 3′

S. pombe cells are plated onto EMM-lysine to select for Lys⁺ transformants. These colonies are pooled and the plasmids are rescued back to E. coli (Hoffman and Winston 1987), selecting for ampicillin-resistance. Individual transformants are checked by plasmid prep and restriction digestion to identify correct plasmids that carry the PDE gene in place of LEU2.

The cloned PDE is then stably introduced into the S. pombe genome by linearizing the plasmid within the lys2 gene on the plasmid and transforming a lys2-97 mutant strain (such as CHP1077) to Lys⁺. By linearizing the plasmid, integration by homologous recombination is greatly enhanced. One can find stable integrants by passaging the Lys⁺ transformants two or three times on nonselective medium (yeast extract agar; this can be done by simply replica plating) and then replica plating back to EMM-lysine medium. The stable Lys⁺ transformants (containing the plasmid integrated at the lys2 locus) will show solid growth on the EMM-Lys plate indicating that most of the cells retain the plasmid, while the original Lys⁺ transformants that did not have the plasmid integrated will show patchy growth, if any, on the EMM-Lys plate due to the high frequency of plasmid loss.

Once a strain carrying the integrated plasmid has been identified, screening strains are constructed by standard genetic crosses as described for the strains expressing PDE genes at the cgs2 locus.

The human PDE10A described in Example 5 herein, has also been put onto the plasmid to express it from the SV40 promoter using SEQ ID NOs:29 and 30. A resulting S. pombe transformant has been identified that has the plasmid integrated into the lys2 locus as described above.

REFERENCES FOR EXAMPLE 6

-   1 Hoffman, C. S. and F. Winston (1987). Gene 57(2-3): 267-72. -   2 Hoffman, R. L. and C. S. Hoffman (2006). Curr Genet 49(6): 414-20. -   3 Wang, L., R. Kao, et al. (2004). Methods 33: 199-205.

Example 7 In Vivo Assay of BC54 Action

A 7 day experiment was conducted to determine the effect of BC54 on mice having transplanted tumors (lymphomas). Drugs were given i.p. once a day in 60-100 ul of PBS.

-   -   3 animals got just BC54—to assess toxicity     -   3 animals injected with lymphoma and treated with BC54     -   3 animals—no treatment     -   1 animal—CHOP 0.5 MTD

BC54 dose was 3 ul of 100 mM solution per mouse per injection. Roughly compared to the in vitro assay, this dose is ˜2.5 times less than the dose used in in vitro. Mice seem to tolerate the treatment painlessly and there are no major signs of toxicity. After 7 days, the untreated mice were terminally ill. The group treated with BC54 looked better although they do show some clinical signs (hunched posture etc). CHOP-treated animal looked healthy. Tumor load was evaluated in the spleen and in the peritoneal lymph nodes (FIG. 7). BC54 reduces the tumor load in lymph nodes of treated mice who have received a transplanted tumor.

Example 8 IL2 Secretion by Concavalin a Treated Jurkat Cells

Jurkat cells, a human T cell lymphoblast-like cell line, increase expression of the cytokine IL2 in response to stimulation by concanavalin A (conA). This effect can be inhibited by elevated cAMP levels, although PDE4 inhibition alone appears to be insufficient to prevent IL2 induction (Kaminuma et al., Immunopharmacology, 1998 January; 38(3):247-52). However, the anti-inflammatory effect of PDE4 inhibitors can be detected using this assay by their ability to enhance the reduction of IL2 secretion by prostaglandin E2. IL2 levels are measured in Jurkat cells (5×10⁵ cells/ml) stimulated by 10 μg/ml ConA in the presence of prostaglandin E2, with or without each test compound (10 mM). After incubation at 37° C. for 24 hours, the resulting supernatant were collected and kept frozen at −70° C. until the time of assay. IL2 in the supernatants were measured by ELISA and the data is presented as a percentage of the PGE2 treated culture (FIG. 12).

All patents, patent applications, and published references cited herein are hereby incorporated by reference in their entirety. It will be appreciated that various of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Various presently unforeseen or unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.

TABLE 7 Structures of BC58 and ten related analogs, together with the effects of these compounds in in vitro enzyme assays. The values represent the percent of PDE activity that is present in the reaction containinq 2 micromolar compound. Structure

Number 58 58-1 PDE4A 31 84 PDE4B 16 64 PDE4D 31 90 PDE7A 68 98 Structure

Number 58-2 58-3 PDE4A 26 14 PDE4B 39 15 PDE4D 30 30 PDE7A 88 20 Structure

Number 58-4 58-5 PDE4A 26 69 PDE4B 47 46 PDE4D 54 68 PDE7A 74 90 Structure

Number 58-6 58-7 PDE4A 73 66 PDE4B 61 57 PDE4D 68 67 PDE7A 75 69 Structure

Number 58-8 58-9 PDE4A 84 84 PDE4B 56 72 PDE4D 76 77 PDE7A 48 52 Structure

Number 58-10 PDE4A 60 PDE4B 39 PDE4D 70 PDE7A 84

TABLE 8 Summary Of Compound Properties Upper panel - ED50 values from yeast 5FOA growth assays. The concentrations of compound required to produce an OD600 of 0.6 is given (in micromolar concentration). Lower panel - IC50 values of the same compound as judged by in vitro enzyme assays using E. coli expressed and purified enzymes. ED₅₀* for 5FOA growth assays Compound PDE4A1 PDE4B2 PDE4D2 PDE4D3 PDE7A PDE7B BC33 23 (m) 28 (r) 1.7 (m) ND 33 >200 ND BC44 ND ND 140 3.3 ND 2.1 FP58 2.1 2.8 >200 >200 >200 >200 Rolipram 13 2 0.5 0.55 >200 >200 FP12 >200 >200 ND ND 2.7 7.8 FP28 >200 45 (m) >200 >200 23 1.5 BC64 20 (m) >200 >200 >200 >200 >200 BRL50481 ND ND ND ND 1.3 42 FP54 0.1 0.1 0.7 13 2.4 0.5 *[Compound] in μM to bring OD₆₀₀ to 0.6 (50% saturated culture). Growth is due to PDE inhibition. IC₅₀ (or AC₂₀₀) Compound PDE4A10 PDE4B2 PDE4D2-CAT^(a) PDE4D2-FL^(b) PDE7A PDE7B Rolipram 1 1 1 <<2 >>2 BRL50481 >>20 0.2 18 12 >>2 >>2 0.5 >>2 1 0.1 28 >10 >10 >2 0.65 1.5 30 >>2 ~2 <0.1 >>2 1 12 54 0.09 0.05 0.11 <<2 0.14 0.14 58 2 1.5 <<2 2 >2 >2 Values represent micromolar (μM) concentrations. AC₂₀₀ [Compound] that doubles PDE activity (cAMP hydrolysis equals that of a reaction containing twice as much enzyme. ^(a)Catalytic fragment of PDE4D2 (residues 86-413) ^(b)Full-length PDE4D2 (residues 1-507)

TABLE 9 Summary of the effect of 2 micromolar compound from the BC12 and BC30 series on PDE7B or PDE4D enzymes. BC12, BC12-1, BC30 and BC30-3 are potent activators of PDE4D. In vitro Inhibition or Activation of PDE7B Compound % Activity 2 uM cpm BC12 149 2388 BC12-1 147 2786 BC12-2 102 10353 BC12-3 92 12032 BC12-4 99 10720 BC58 73 15093 DMSO 100 10618 No enzyme 0 27339 In vitro activation of PDE4D catalytic domain Compound % activity 2 uM cpm BC12 133 3686 BC12-1 137 2965 BC12-2 99 10223 BC12-3 88 12387 BC12-4 103 9372 BC30 139 2583 BC30-1 75 14743 BC30-2 115 7133 BC30-3 130 4314 BC30-4 105 9003 DMSO 100 10029 No enzyme 0 29182

TABLE 10 3 HOURS 20 HOURS PDE4 PDE7 TNF, % EtOH % EtOH inhibitor inhibitor pg/ml cntrl TNF, pg/ml cntrl — — 4926 102.0 13513 98.7 — 30 3317 68.7 7481 54.7 35 — 4383 90.7 11728 85.7 35 30 2778 57.5 4051 29.6 — low BRL 4491 93.0 12718 92.9 — high BRL 4565 94.5 13111 95.8 rolipram — 3497 72.4 9362 68.4 rolipram low BRL 3633 75.2 10228 74.7 rolipram high BRL 3573 74.0 9572 69.9 58 — 3345 69.2 10030 73.3 58 30 2279 47.2 4258 31.1 — — 4737 98.0 13856 101.3 Conclusions: 1) BC58 mimics BC35 and rolipram as a PDE4 inhibitor with anti-inflammatory properties. 2) BC30 (a PDE7 inhibitor) shows superior synergy for reducing TNF alpha release by LPS-treated U937 cells to that of BRL50481, when combined with our PDE4 inhibitors BC35 or BC58. TNF alpha is an inflammtory response that is commonly used to assess the effectivesness of PDE inhibitors.

TABLE 11 Conclusions: 1) BC27, BC35, and BC58 all act like PDE4 inhibitors with moderate TNF alpha reduction on their own and potent synergy with the PDE7 inhibitor BC30. 10 uM cmpd, % vehicle control [BC30], uM after 3 h. after 20 h. EtOH + DMSO 99.9 105.6 58/0 61.2 53.8 58/2 46.2 36.8 58/10 33.3 16.4 27/0 75.3 78.3 27/2 54.8 41.1 27/10 31.8 15.0 EtOH + DMSO 100.3 94.4 35/0 95.8 66.1 35/2 66.9 35.4 35/10 45.6 19.4 no LPS 2.3 1.2 Compounds 58, 27 and 35 are at 10 uM Compound 30 is at either 0, 2 or 10 uM 58/2 means 10 uM cmpd 58, 2 uM 30, etc.

TABLE 12 Conclusions: BC12 shows potent synergy with rolipram for reduction of TNF alpha Treatment 3 hours 20 hours EtoH 100.0 100.0 Rolipram 86.2 86.3 BRL 92.5 102.6 12 42.5 102.3 Roli + BRL 71.7 82.0 Roli + 12 14.0 8.5 BRL + 12 19.2 61.7 Roli + BRL + 12 14.5 9.3

TABLE 13 Structures and Composite Z scores for 115 screened compounds. These compounds all possess the core structure found in BC58, BC33, BC44, BC28, and BC64. Nearly half of these compounds produce Composite Z scores greater than 8.53, which is considered a statistically-significant inhibitor by the Broad screening computationalists. PDE4A1 PDE4A5 PDE4B3 PDE7A BC ID Image Plate/well SMILE 50.594 36.026 42.416 −0.794 BC33

2012L11 CCC1CCc2c(C1)sc3ncnc(N4CCOCC4)c23 22.267 63.222 85.067 177.31 BC44

2051L22 CCN(CC)c1ncnc2sc3CCCCc3c12 34.335 154.72 78.927 24.881 BC58

2040L10 CC1CCN(CC1)c2ncnc3sc(C(═O)Nc4ccc(F)cc4F)c(C)c23 27.41 NaN NaN NaN BC58-3

1159O15 COc1cc(NC(═O)c2sc3ncnc(N4CCCCC4C)c3c2C)cc(OC)c1OC 28.238 NaN NaN NaN BC58-7

1159C17 CCN(CC)c1ncnc2sc(C(═O)Nc3cccc(OC)c3)c(C)c12 25.972 111.95 78.662 123.57 BC58-9

2040B18 CN(c1ccccc1)c2ncnc3sc(C(═O)Nc4ccc(F)cc4F)c(C)c23 32.338 NaN NaN NaN BC58-10

1159A21 Cc1c(sc2ncnc(N3CCc4ccccc4C3)c12)C(═O)Nc5ccc6OCCOc6c5 24.925 NaN 0.3426 55.035 BC64

2086I09 Cc1sc2ncnc(Nc3ccccc3)c2c1C 32.257 20.422 43.053 26.421

2012B19 COc1ccc(CCNc2ncnc3sc4CCCCc4c23)cc1 34.225 26.878 35.319 30.877

2012F09 Fc1ccc(cc1)C(═O)Nc2ncnc3sc4CCCCCc4c23 1.6797 NaN 9.3964 17.695

2077P12 CC(C)CNc1ncnc2sc3CCCCc3c12 29.047 NaN 0.593 18.891

2084 E21 C1CCC(C1)Nc2ncnc3sc4CCCc4c23 7.8022 NaN 0.9421 12.419

2084I06 O═C(Nc1ncnc2sc3CCCc3c12)c4cccs4 11.225 1.7466 16.5 −1.481

2040F10 Cc1c(sc2nc(C)nc(N3CCCCC3)c12)C(═O)Nc4cccc(C4)C(F)(F)F 10.127 NaN 25.393 NaN

1426G17 Cc1sc2ncnc(NCCc3ccc4OCCOc4c3)c2c1C 11.659 10.539 −0.456 −1.377

2041K04 CCC(C)NC(═O)c1sc2nc(C)nc(N(C)c3ccc(cc3)C(C)C)c2c1C 19.619 33.878 4.5126 0.4852

2041K21 COc1cccc(NC(═O)c2sc3ncnc(N4CCc5ccccc5C4)c3c2C)c1 0.6949 13.196 0.2682 0.2354

2007L04 CCOC(═O)c1sc2nc(C)nc(N(C)Cc3ccccc3)c2c1C 26.235 NaN NaN NaN

1159I19 Cc1c(sc2ncnc(N3CCc4ccccc4C3)c12)C(═O)NCc5ccco5 17.363 NaN NaN NaN

1159M15 COc1cc(NC(═O)c2sc3nc(C)nc(N4CCCCC4C)c3c2C)cc(OC)c1OC 11.945 NaN NaN NaN

1159O19 Cc1c(sc2ncnc(N3CCc4ccccc4C3)c12)C(═O)NCc5ccccn5 20.429 NaN −0.752 NaN

1380G04 CCN(CC)c1ncnc2sc3CC(CCc3c12)(C#N)c4ccccc4 36.663 NaN −0.935 NaN

1380I07 O═C(Nc1ncnc2sc3CCCc3c12)c4ccccc4 10.795 NaN 0.7173 NaN

1392J18 COC(═O)Cc1nc(N2CCC(C)CC2)c3c4CCCCc4sc3n1 12.819 NaN 0.0445 NaN

1407G21 CC(C)N(C)c1ncnc2sc3CCCc3c12 9.1375 NaN −0.382 NaN

1414F18 COc1ccc(cc1OC)C(═O)Nc2ncnc3sc4CCCCc4c23 11.67 NaN −1.188 −1.925

2074G03 Cc1sc2ncnc(N3CCN(CC3)c4cccc(c4)C(F)(F)F)c2c1C 18.511 NaN 5.6256 0.8496

2083J20 O═C(N1CCN(CC1)c2ncnc3sc4CCCCc4c23)c5ccco5 14.925 3.411 −1.056 1.3549

2040H10 CC1CCCCN1c2ncnc3sc(C(═O)NCCC4═CCCCC4)c(C)c23 8.6742 0.9752 3.2214 0.6002

2040L12 CCN(CC)c1nc(C)nc2sc(C(═O)Nc3cc(C1)ccc3OC)c(C)c12 12.024 1.3723 3.1975 0.7021

2040N10 CC1CCN(CC1)c2nc(C)nc3sc(C(═O)N4CCN(CC4)c5ccc(F)cc5)c(C)c23 12.157 1.3091 1.0041 1.3653

2040P12 CCOC(═O)c1ccccc1NC(═O)c2sc3nc(C)nc(N(CC)CC)c3c2C 7.3705 −0.705 12.239 −1.037

2040N12 CCN(CC)c1nc(C)nc2sc(C(═O)N3CCCCCC3)c(C)c12 −0.629 0.7964 33.162 −1.371

2041G21 CN(Cc1ccccc1)C(═O)c2sc3ncnc(N4CCN(CC4)c5ccccc5)c3c2C 3.7863 −0.22 9.0572 0.7038

2053H16 CCNc1nc(SCC(═O)OCC)nc2sc3CC(C)CCc3c12 1.6456 NaN 21.25 NaN

1414C02 CN(C)CCCNc1ncnc2sc3CCCCc3c12 3.4896 NaN 21.717 NaN

1424K08 Cc1c(sc2nc(C)nc(N3CCOCC3)c12)C(═O)Nc4cccc(O)c4C 8.4736 NaN 31.009 NaN

1435C20 C═CCNc1ncnc2sc3CCCCc3c12 6.7542 NaN 47.874 NaN

1439G17 CC1CCc2c(C1)sc3nc(SCC(═O)N)nc(NCc4ccccc4)c23 2.3434 NaN NaN NaN

1157L22 CCOC(═O)c1sc2nc(CC(═O)OC)nc(N3CCN(CC3)c4ccc(OC)cc4)c2c1C 0.3744 NaN NaN NaN

1158A03 CCOC(═O)c1sc2nc(CC(═O)OC)nc(NCCN3CCOCC3)c2c1C 2.6937 NaN NaN NaN

1158C03 CCN(CC)CCCNC(═O)c1sc2ncnc(N3CCN(CC3)c4ccc(OC)cc4)c2c1C −0.078 NaN NaN NaN

1158G03 CN1CCN(CC1)c2nc(C)nc3sc(C(═O)N4CCN(CC4)c5cccc(Cl)c5)c(C)c23 2.4477 NaN NaN NaN

1159A19 CCOC(═O)CNC(═O)c1sc2ncnc(N3CCN(CC3)c4ccccc4F)c2c1C 2.6846 NaN NaN NaN

1159C19 COCCCNC(═O)c1sc2ncnc(N3CCN(CC3)c4ccccc4F)c2c1C 3.9976 NaN NaN NaN

1159C21 Cc1c(sc2ncnc(N3CCc4ccccc4C3)c12)C(═O)Nc5ccc6OCOc6c5 1.0613 NaN NaN NaN

1159 E19 Cc1c(sc2ncnc(N3CCN(CC3)c4ccccc4)c12)C(═O)NCC5CCCO5 5.3411 NaN NaN NaN

1159G15 CCOC(═O)c1sc2ncnc(N3CCC4(CC3)OCCO4)c2c1C 7.7398 NaN NaN NaN

1159G19 Cc1c(sc2ncnc(N3CCN(CC3)c4ccccc4)c12)C(═O)NCc5ccco5 0.5937 NaN NaN NaN

1159K09 COC(═O)c1ccccc1NC(═O)c2sc3ncnc(N4CCN(CC4)c5ccccn5)c3c2C 5.4376 NaN NaN NaN

1159K19 CCCCNC(═O)c1sc2ncnc(N3CCc4ccccc4C3)c2c1C 1.1811 NaN NaN NaN

1159M09 COCCNC(═O)c1sc2ncnc(N3CCN(CC3)c4ccccn4)c2c1C 1.9801 NaN NaN NaN

1159M17 Cc1c(sc2ncnc(N3CCN(CC3)c4ccccc4F)c12)C(═O)NCC5CCCO5 5.0576 NaN NaN NaN

1159M19 COCCCNC(═O)c1sc2ncnc(N3CCc4ccccc4C3)c2c1C 0.8757 NaN NaN NaN

1159O09 COc1cc(NC(═O)c2sc3nc(C)nc(N4CCOCC4)c3c2C)cc(OC)c1OC 3.616 NaN NaN NaN

1159O17 Cc1c(sc2ncnc(N3CCN(CC3)c4ccccc4F)c12)C(═O)NCc5ccco5 −0.126 NaN 3.0462 NaN

1379F18 CCOC(═O)c1sc2nc(CC(═O)OC)nc(NCC(═O)OC)c2c1C −0.753 NaN 1.8207 NaN

1379L18 CCOC(═O)c1sc2ncnc(N3CCc4ccccc4C3)c2c1C 1.5734 NaN −0.703 NaN

1379O22 CC(C)(C)C(═O)Nc1ncnc2sc3CCCCc3c12 1.2506 NaN −1.076 NaN

1380I09 N#CC1(CCc2c(C1)sc3ncnc(N4CCCC4)c23)c5ccccc5 −1.217 NaN −2.398 NaN

1380M03 CCC(CO)Nc1nc(C)nc2sc3CCCc3c12 1.3339 NaN 0.2365 NaN

1381A19 CC(C)C(Nc1ncnc2sc3CCCc3c12)C(═O)O −0.467 NaN 0.3352 NaN

1381O17 OC(═O)C(Cc1ccccc1)Nc2ncnc3sc4CCCc4c23 0.1027 NaN 0.4676 NaN

1382J15 CNc1ncnc2sc(C)c(C)c12 3.1682 NaN −1.206 NaN

1393 E07 CN(c1ccccc1)c2ncnc3sc(C(═O)N4CCC5(CC4)OCCO5)c(C)c23 1.647 NaN 0.0332 NaN

1403J21 CCN(CC)c1ncnc2sc(C)c(C)c12 0.1456 NaN 0.5128 NaN

1406P09 FC(F)(F)C(═O)Nc1ncnc2sc3CCCCc3c12 2.9506 NaN 2.8289 NaN

1410G03 CCNc1nc(C)nc2sc3CCCCc3c12 1.6242 NaN 0.632 NaN

1414C06 CCN1CCN(CC1)c2nc(C)nc3sc4CCCc4c23 0.327 NaN 0.6059 NaN

1415C05 CN1CCCN(CC1)c2nc(C)nc3sc4CCCc4c23 2.2076 NaN 0.8257 NaN

1415 E01 OCCN(CCO)c1ncnc2sc3CCCCc3c12 1.7784 NaN 1.1976 NaN

1415M01 COc1ccccc1C(═O)Nc2ncnc3sc4CCCCc4c23 4.6254 NaN 4.7765 NaN

1421A20 C1Cc2sc3ncnc(N4CCCCC4)c3c2C1 2.1932 NaN 5.4669 NaN

1421B05 CC1CN(CC(C)O1)c2nc(C)nc3sc4CCCc4c23 0.1866 NaN −0.936 NaN

1422J17 CCC(CO)Nc1nc(C)nc2sc3CCCc3c12 0.9043 NaN 4.2065 NaN

1423F01 COc1ccc(CCNc2ncnc3sc(C)c(C)c23)cc1OC 1.7161 NaN 0.501 NaN

1423I22 C(Nc1ncnc2sc3CCCCc3c12)c4cccnc4 2.2036 NaN −2.898 NaN

1424A12 CC1CCN(CC1)c2nc(C)nc3sc(C(═O)N4CCOCC4)c(C)c23 0.4867 NaN −1.074 NaN

1425N16 CC1CCc2c(C1)sc3ncnc(NCCN4CCOCC4)c23 1.5745 NaN 0.9419 NaN

1426A15 Cc1sc2ncnc(NCc3ccc4OCOc4c3)c2c1C −0.147 NaN −0.516 NaN

1426C09 Cc1sc2ncnc(NC3CCN(Cc4ccccc4)CC3)c2c1C −0.033 NaN −0.071 NaN

1426I13 Cc1nc(NCc2ccco2)c3c4CCCc4sc3n1 0.4682 NaN 0.0693 NaN

1426O11 CCC(CO)Nc1nc(C)nc2sc3CCCCc3c12 1.6661 NaN 0.5336 NaN

1435 E20 C(CNc1ncnc2sc3CCCCc3c12)Cn4ccnc4 0.5702 NaN 1.2391 NaN

1439B09 CCOC(═O)CSc1nc(NC)c2c3CCCCc3sc2n1 −1.628 −0.574 0.0838 −0.398

2005M12 C(Cc1cccs1)Nc2ncnc3sc4CCCc4c23 −0.396 −10.3 0.2826 −0.759

2007D06 CCOC(═O)c1sc2nc(C)nc(N3CCC(C)CC3)c2c1C 0.0978 −10.26 0.3145 1.5462

2007H06 CCCCN(CC)C(═O)c1sc2ncnc(N3CCN(CC3)c4ccc(OC)cc4)c2c1C 0.1429 −4.881 0.0156 0.03

2008G09 Cc1nc(NCCc2ccccc2)c3c4CCCCc4sc3n1 −1.395 −4.329 0.0615 0.9074

2008K11 O═C(Nc1ncnc2sc3CCCc3c12)/C═C/c4ccccc4 0.407 −0.102 0.1698 −0.757

2012H20 CC(C)CC(Nc1ncnc2sc3CCCc3c12)C(═O)O 1.67 −0.334 −0.543 −0.684

2012L13 Cc1cc(C)c(C)c(c1C)S(═O)(═O)N2CCN(CC2)c3ncnc4sc5CCCCc5c34 2.8 0.289 −0.716 1.8846

2012O16 OC(═O)CCNc1ncnc2sc3CCCCc3c12 3.1355 −0.897 4.66 −0.593

2040D04 CCOC(CNc1nc(C)nc2sc3CCCCc3c12)OCC 3.8961 2.5342 5.0875 1.5898

2040F07 CCOC(═O)c1sc2nc(Cc3ccccc3)nc(NCCC4═CCCCC4)c2c1C 3.5005 −0.722 0.9277 −0.598

2040F13 CC(C)CCNC(═O)c1sc2ncnc(N3CCOCC3)c2c1C 3.0133 0.4891 1.7278 0.6002

2040J10 COc1cc(OC)c(NC(═O)c2sc3ncnc(N4CCCCC4C)c3c2C)cc1Cl 7.0857 −0.227 5.3667 −0.165

2040J12 CCN(CC)c1ncnc2sc(C(═O)N(C)C3CCCCC3)c(C)c12 1.9156 2.4446 −0.408 0.5923

2041 E21 COc1ccccc1CNC(═O)c2sc3ncnc(N4CCN(CC4)c5ccccc5F)c3c2C 5.2983 1.1627 −0.545 0.7201

2041I04 COc1ccc(NC(═O)c2sc3nc(C)nc(N(C)c4ccc(cc4)C(C)C)c3c2C)c(OC)c1 0.2744 −0.939 7.48 −0.933

2041I21 Cc1c(sc2ncnc(N3CCN(CC3)c4ccccc4)c12)C(═O)N5CCCCCC5 2.4965 2.0846 0.936 −0.346

2051H22 OCCNc1ncnc2sc3CCCCc3c12 1.1862 −4.733 −0.998 −0.445

2051N22 C(CNc1ncnc2sc3CCCCc3c12)CN4CCOCC4 −1.088 −0.378 0.2798 −0.995

2054 E08 CNc1nc(═S)[nH]c2sc3CCCCc3c12 0.2389 0.5516 0.62 −0.737

2054 E16 CC1═NN(C(═O)C1)c2ncnc3sc4CCCCc4c23 −0.307 1.0728 0.0285 0.1836

2054N05 CCNc1nc(SCC(═O)NC(═O)NCc2ccccc2)nc3sc4CN(C)CCc4c13 2.3863 1.0025 −0.211 1.181

2054P05 CCNc1nc(SCc2ccc(C1)cc2)nc3sc4CN(C)CCc4c13 −0.348 NaN 0.8366 0.7527

2074A07 C(N1CCN(CC1)c2ncnc3sc4CCCCCc4c23)c5ccccc5 1.1575 NaN 1.9013 1.1908

2077J03 C(Nc1ncnc2sc3CCCCc3c12)c4ccccc4 0.1374 NaN 1.6993 0.439

2081F17 Cc1sc2ncnc(NCc3cccnc3)c2c1C 1.9931 NaN −0.877 −1.18

2083L15 CCN(CC)CCCNc1ncnc2sc3CCC(C)Cc3c12 0.1942 NaN 0.6718 0.0079

2084A21 Cc1sc2ncnc(N3CCN(CCO)CC3)c2c1C 6.4203 NaN 0.884 −1.808

2084G06 O═C(Nc1ncnc2sc3CCCCCc3c12)c4ccco4 0.3168 NaN 0.8136 −1.774

2085F18 Cc1sc2ncnc(NCC(═O)O)c2c1C 1.0991 NaN −1.519 −0.627

2085O15 CN(C)c1ncnc2sc(C)c(C)c12

TABLE 14 Compounds disclosed in the published PCT application WO 2008/130619. Formula I (Group I)

wherein X is SO, or SO₂, R1 is H, or alkyl, R2 is alkyl, or halogen. In specific embodiments, R1 is Me. In other specific embodiments R1 is F. In certain embodiments R2 is t-Bu. In specific embodiments, R1 is methyl. In more specific embodiments, the compounds are selected from:

(I-1 to I-5). Formula (II) (Group II):

wherein R1 is alkyl, R2 is aryl or heteroaryl, R3 is alkyl, aryl, cycloakyl, or alkylaryl. In specific embodiments, R1 is methyl. In certain embodiments R2 is furanyl or thiophenyl. In other specific embodiments, R2 is substituted phenyl or benzyl. In preferred embodiments, R3 is iso-butyl. In more specific embodiments, the compounds

are selected from:

(II-1 to II-13) Formula III (Group III):

wherein R1 is nitrile, or alkylcarboxylate, R2 is alkyl, aryl, or heteroaryl. In specific embodiments, R1 is nitrile or methylcarboxylate. In certain embodiments, R2 is a five membered heteroaryl. In more specific embodiments, R2 is furanyl, or thienyl. In other embodiments, R2 is a six membered aryl. In more specific embodiments, R2 is substituted phenyl.

(III-1 to III-5) Formula IV (Group IV):

wherein R1 is alkyl, alkenyl, or alkylcarboxylicacid, R2 is halogen. In certain embodiments R1 is butyl. In other embodiments R1 is terminal alkenyl. In more specific embodiments R1 is allyl, or vinyl. In other embodiments, R1 is C₁₋₄alkyl. In specific embodiments R1 is methylcarboxylicacid. In certain embodiments R2 is Cl, or Br. In more specific embodiments, the compounds are selected from:

(IV-1 to IV-14) Formula V (Group V):

wherein R1 is CO, or alkylalcohol, R2 is alkyl, R3 is alkoxy, and the C4 and C9 stereocenters are independently (R) or (S). In certain embodiments R1 is carbonyl, or 2-methylpropan-1-ol. In specific embodiments R2 is methyl. In certain embodiments, R3 is methoxy. In more specific embodiments the compounds are selected from:

(V-1 to V-4) Formula VI (Group VI)

wherein R1 is hydrogen, hydroxyl, carbonyl, or alkylalcohol, R2 and R3 are independently selected from hydrogen, alkyl, alkylcarboxylate, or carboxylic acid, R4 is hydrogen, or alkyl, R5 is hydrogen, alkyl, hydroxyl, or acetate, R6 is hydrogen, or alkoxy, and the C4 and C9 stereocenters are independently (R) or (S). In certain embodiments R1 is 2-methylpropan-1-ol. In specific embodiments R2 is methyl. In certain embodiments, R2 is methylcarboxylate. In specific embodiments R2 and R3 are both methyl. In other embodiments, R2 is methyl, and R3 is methylcarboxylate. In specific embodiments R4 is iso-propyl. In specific embodiments, R5 is methyl. In certain embodiments, R6 is methoxy. In more specific embodiments the compounds are selected from:

(VI-1 to VI-4) These are referred to as Group VI.

TABLE 15 Compound PDEs Alias Structure Smiles Activity 4A1 4A5 4B3 4D2 4D3 7A 22 (4)

COC(═O)C/1 = C(C)N(C2CCCCC2)C(═O)\C1 = C/c3ccc3 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response (% rolipram) IC50  98.84  27.01  9.42  54.00   ND −0.06 13.66  1.27 ND   ND 70.26 37.54  0.14 51.00   ND ND ND ND 34.00   ND ND 42.32 >200 12.00   ND 132.82  57.22 >200 ND   ND 27 (3)

COC(═O)C/1 = C(C)N(C(═O)\C1 = C/c2ccccs2)c3ccccc3C1 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response (% rolipram) IC50  64.18  17.41 200.00  78.00   100 nM 65.67 12.63 13.00 ND   ND −0.26 22.05  3.80 71.00   ND ND ND ND 11.00   ND ND  0.00 >200  8.00   100 nm  2.34  5.07 >200 ND   ND 27-1 (1)

COC(═O)C/1 = C(C)N(C)C(═O)\C1 = C/c2cccs2 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50 ND ND 107.90 ND ND ND ND  3.41 ND ND ND ND >200 ND ND ND ND >200 ND ND ND ND >200 ND ND ND ND >200 ND ND 27-2 (2)

COC(═C/1 = C(C)N(OC(C)C)C(═O)\C1 = C/c2ccco2 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50 ND ND  4.83 ND ND ND ND  0.75 ND ND ND ND  2.66 ND ND ND ND 30.57 ND ND ND ND  68.40 ND ND ND ND >200 ND ND 27-3 (1)

COC(═C/1 = C(C)N(CC(C)C)C(═O)\C1 = C/c2cccs2 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50 ND ND  7.63 ND ND ND ND  0.85 ND ND ND ND  3.84 ND ND ND ND >200 ND ND ND ND >200 ND ND ND ND >200 ND ND 27-4 (1)

COC(═C/1 = C(C)N(C(═O)\C1 = C/c2cccs2)c3ccccc3 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50 ND ND  27.66 ND ND ND ND  1.33 ND ND ND ND  3.57 ND ND ND ND >200 ND ND ND ND >200 ND ND ND ND >200 ND ND 27-5 (2)

C3 = CSC(C = C2C(═O)N(CC1 = CC = CC(C) = C1)C(C) = C2C(OC)═O) = C3 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50 ND ND  1.01 ND ND ND ND  0.23 ND ND ND ND  2.04 ND ND ND ND 17.15 ND ND ND ND  14.74 ND ND ND ND >200 ND ND 27-6 (4)

C3 = (C = C2C(═O)N(C1 = CC = C(F)C = C1)C(C) = C2C(OC)═O) = C3 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50 ND ND  4.26 ND ND ND ND  0.83 ND ND ND ND  2.34 ND ND ND ND >200 ND ND ND ND >200 ND ND ND ND  13.04 ND ND 27-7 (3)

C3 = (C = C2C(═O)N(C1 = CC = CC(C(F)(F)F) = C1)C(C) = C2C(OC)═O) OC) = C3 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50 ND ND >200   ND ND ND  2.31   ND ND ND >200   ND ND ND >200   ND ND ND >200   ND ND ND >200   ND 27-8 (2)

COC(═C/1 = C(C)N(C(═O)\C1 = C/c2ccco2)c3ccccc3C1 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50 ND ND  2.74 ND ND ND ND  1.55 ND ND ND ND 18.38 ND ND ND ND >200 ND ND ND ND >200 ND ND ND ND >200 ND ND 2008, C21 (2)

COC(═C/1 = C(C)N(C(═O)\C1 = C/c2cccc(OC)c2O)ccccc3F 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50  18.97 ND ND ND ND  1.80 ND ND ND ND 24.16 ND ND ND ND ND ND ND ND ND ND ND ND ND ND  −0.08 ND ND ND ND 2008, K19 (1)

COC(═C1 = C(C)N(/C(═C)/c/c2cccs2)/C1 = O)c3ccccc3 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50  −0.46 ND ND ND ND  0.06 ND ND ND ND −0.61 ND ND ND ND ND ND ND ND ND ND ND ND ND ND  2.43 ND ND ND ND 2008, 019 (1)

COC(═C/1 = C(C)N(C(═O)\C1 = C/c2ccco2)c3ccc(cc3)C(C)C 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50  0.80 ND ND ND ND  1.73 ND ND ND ND −2.99 ND ND ND ND ND ND ND ND ND ND ND ND ND ND  2.78 ND ND ND ND (1) Strong PDE4A5 inhibitor (2) Strong PDE4A, 4B inhibitor; Moderate PDE4D inhibitor (3) Strong PDE4A. 4B inhibitor (4) Strong PDE4A, 4B, PDE7A inhibitor: Weak PDE4D inhibitor

TABLE 16 Compound PDEs Alias Structure Smiles Activity 4A1 4A5 4B3 4D2 4D3 7A 30 KM03472 (2)

Nc1c(C#N)sc2sc(C#N)c(c3ccco3)c12 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50 11.40  4.56 ND 65.7 ND 2.14 2.79 ND ND ND  4.67  5.21 ND 67 ND ND ND ND 7.5 ND ND −0.55 ND   4 ND 103.99  67.85  4 153.6 ND 1441, G04 KM03473 30-2 (1)

Nc1c(C#N)sc2sc(C#N)c(c3cccs3)c12 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50 −0.2539 ND ND ND ND ND ND ND ND ND −0.3999 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND active/plateaus ND ND Maybridge: KM03484 30-4 (3)

CC(C)(C)c1c(C#N)sc2sc(C#N)c(N)c12 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND weak activity ND ND 554, N12 KM03474 30-3 (3)

Nc1c(C#N)sc2sc(C#N)c(c3ccc(Cl)cc3)c12 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50 ND ND ND ND ND ND ND ND ND ND  1.462 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND inactive ND ND 1441, C04 KM03459 30-1 (4)

CCOC(═O)c1sc2sc(C(═O)OCC)c(c3ccco3)c2c1N 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50  0.5758 ND ND ND ND ND ND ND ND ND  8.4406 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND inactive ND ND 2055, C21

COC(═O)c1sc(SC)c(C#N)c1c2ccco2 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50 15.8505 ND ND ND ND 2.07 ND ND ND ND 21.4955 ND ND ND ND ND ND ND ND ND ND ND ND ND ND  0.2998 ND ND ND ND 1449, A02

NC(═O)c1sc(C#N)c(c2ccco2)c1c3ccco3 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50  1.3541 ND ND ND ND ND ND ND ND ND  1.8244 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 2055, N01

CCOC(═O)c1sc2sc(C(═O)OCC)c(c3ccc(Cl)cc3)c2c1N 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50  2.1835 ND ND ND ND 0.582 ND ND ND ND  1.8421 ND ND ND ND ND ND ND ND ND ND ND ND ND ND  1.4276 ND ND ND ND (1) Weak PDE7A inhibitor (2) Strong PDE7A inhibitor (3) Lillte/no activity under conditions used (4) Weak PDE4A inhibitor

TABLE 17 Compound PDEs Alias Structure Smiles Activity 4A1 4A5 4B3 4D2 4D3 7A 35 (1)

CC(C)n1c2ccc(Cl)cc2c3nc4ccccc4nc13 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50  78.02  9.44  5.80  96 100 nM 66.33 11.29 >200 ND nl ND  0.17  11.39 >200  76 ND ND ND >200 10 ND ND  −0.03 >200  3  35 nM −0.61 −0.37 >200 ND ND 41 (1)

CC(C)n1c2ccc(C)cc2c3nc4ccccc4nc13 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50 106.90 ND  3.50 ND 650 nM 84.08 ND  0.40 ND ND 200.51 ND  18.00 ND ND ND ND >200 ND ND ND ND >200 ND 200 nM  0.31 ND >200 ND ND 1422, G02 (1)

Ccn1c2ccccc2c3nc4ccccc4nc13 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50  70.89 ND ND ND ND ND ND ND ND ND 103.69 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 1422, I02 (1)

CC(C)n1c2ccccc2c3nc4ccccc4nc13 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50  56.62 ND ND ND ND ND ND ND ND ND  99.04 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 1422, K02 (1)

CCCn1c2ccccc2c3nc4ccccc4nc13 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50  72.21 ND ND ND ND ND ND ND ND ND 115.27 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 1422, M02 (2)

CC(C)Cn1c2ccccc2c3nc4ccccc4nc13 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50  40.53 ND ND ND ND ND ND ND ND ND  86.04 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 1427, P20 (3)

C = CCn1c2ccccc2c3nc4ccccc4nc13 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50  31.38 ND ND ND ND ND ND ND ND ND 103.46 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 1429, L06 (4)

OC(═O)Cn1c2ccc(Cl)cc2c3nc4ccccc4nc13 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50  29.15 ND ND ND ND ND ND ND ND ND  41.77 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 2013, I20 (1)

CCCCn1c2ccccc2c3nc4ccccc4nc13 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50  80.55 ND ND ND ND 71.82 ND ND ND ND  0.29 ND ND ND ND ND ND ND ND ND ND ND ND ND ND −0.84 ND ND ND ND 2015, L04 (1)

CC(C)Cn1c2cccBrcc2c3nc4ccccc4nc13 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50  13.51 ND ND ND ND 57.60 ND ND ND ND  1.17 ND ND ND ND ND ND ND ND ND ND ND ND ND ND −0.79 ND ND ND ND 2015, P06 (4)

CCc1ccc2n(CC)c3nc4ccccc4nc3c2c1 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50  23.52 ND ND ND ND 51.82 ND ND ND ND  0.13 ND ND ND ND ND ND ND ND ND ND ND ND ND ND  1.07 ND ND ND ND 2017, C18 (4)

Brc1ccc2n(CC = C)c3nc4ccccc4nc3c2c1 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50  49.17 ND ND ND ND 69.70 ND ND ND ND  56.91 ND ND ND ND ND ND ND ND ND ND ND ND ND ND −1.58 ND ND ND ND 2019, G03 (4)

c(cn1c2ccccc2c3nc4ccccc4nc13)c5ccccc5 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50  36.37 ND ND ND ND 49.61 ND ND ND ND  83.22 ND ND ND ND ND ND ND ND ND ND ND ND ND ND  4.47 ND ND ND ND 2033, H06 (4)

C = Cn1c2ccccc2c3nc4ccccc4nc13 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50  29.62 ND ND ND ND 66.58 ND ND ND ND 103.11 ND ND ND ND ND ND ND ND ND ND ND ND ND ND  0.63 ND ND ND ND (1) Strong PDE4A, PDE4B inhibitor (2) Strong PDE4A inhibitor (3) Strong PDE4B, moderate PDE4A inhibitor (4) Moderate PDE4A, PDE4B inhibitor (5) Moderate PDE4B inhibitor

TABLE 18 Compound PDEs Alias Structure Smiles Activity 4A1 4A5 4B3 4D2 4D3 7A 39 (1)

COc1ccc2CC[C@@H]3CC(═O)CC[C@]3(C)c2c1 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50 −0.53 ND >200 68.10 ND 20.53 ND >200  38.30 ND 25.84 ND 51.75 37.50 ND ND ND >200 8.50 ND −0.74 ND >200 4.10 ND 135.37 ND  5.77  55.30 ND 2158J12 (2)

Cc1ccc2c(CC[C@H]3CC(═O)CC[C@@]32C)c1 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50 81.97  2.18 ND ND ND 113.27  7.00 ND ND ND 37.13  8.18 ND ND ND ND ND ND ND ND 14.84 40.17 ND ND ND  74.45  29.01 ND ND ND 11 (3)

COc1ccc2CC[C@H]3CC(═O)CC[C@]3(C)c2c1 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response (% BRL50481) IC50 −0.01 ND ND ND   ND  −0.32 ND ND ND   ND  −2.22 ND ND ND   ND ND ND ND ND   ND −0.58 ND ND ND   ND 88.24 ND  22.00  89.00   ND 2067M03 (1)

COc1ccc2CCC3[C@@](C)(CO))CCC[C@]3(C)c2c1 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response IC50 11567  6.63 ND ND ND  16.94  8.43 ND ND ND 13.34 19.00 ND ND ND ND ND ND ND ND ND  1.24 ND ND ND 104.31  8.65 ND ND ND (1) Strong PDE7A inhibitor, weak to moderate PDE4A, 4B inhibitor (2) Strong PDE7A and PDE4s (including PDE4D) inhibitor (3) Strong PDE7A inhibitor

TABLE 19 Compound Alias Structure Smiles Activity 4A1 26

Cc1cc(C)nc(SCCS(═O)(═O)c2ccc(cc2)C(C)(C)C)n1 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response (% rolipram) IC50  99.87  26.63  2.44  96.00   500 nM 1428p07

Cc1cc(C)nc(SCCS(═O)c2ccc(cc2)C(C)(C)C)n1 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response (% rolipram) IC50  53.44 ND ND ND   ND 1437 E15

Cc1ccc(cc1)S(═O)(═O)CCSc2ncccn2 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response (% rolipram) IC50 −0.15 ND ND ND   ND 1437C15

Fc1ccc(cc1)S(═O)(═O)CCSc2ncccn2 1 Assay (20 uM) 2 Assay (2 uM) ED50 cAMP Response (% rolipram) IC50  0.65 ND ND ND   ND (1) Strong PDE4A, 4B inhibitor (2) Moderate 4A inhibitor

PDEs 4A5 4B3 4D2 4D3 7A 70.97 0.39 ND ND 0.58 13.13 12.54 ND 0.97 0.13 0.24 0.13 ND >200 >200 57.30 92.00 17.00 14.00 27.90 ND ND ND 500 nm ND ND 103.15 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 3.19 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 4.18 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 

1. A composition comprising (a) a phosphodiesterase (PDE) inhibitor represented by any one of the formulas of groups NI to NIII, wherein the PDE inhibitor inhibits PDE4 activity, PDE7 activity or both PDE4 activity and PDE7 activity and (b) a pharmaceutically acceptable carrier.
 2. The composition of claim 1, wherein the composition specifically inhibits PDE4A activity, PDE4B activity and/or PDE7A activity.
 3. A composition comprising (a) a phosphodiesterase (PDE) inhibitor represented by any one of the formulas of groups NI to NIII, wherein the PDE inhibitor inhibits PDE4 activity, PDE7 activity or both PDE4 activity and PDE7 activity, (b) a phosphodiesterase (PDE) inhibitor represented by any one of the formulas of groups I to VI as shown in Table 8, wherein the PDE inhibitor inhibits PDE4 activity, PDE7 activity or both PDE4 activity and PDE7 activity, and (c) a pharmaceutically acceptable carrier.
 4. A method for treating a PDE-associated disease or condition in an individual, comprising: administering to an individual in need thereof a therapeutically effective amount of a composition of claim 1 to treat the PDE-associated disease or condition in the subject.
 5. The method of claim 4, wherein the individual is human.
 6. The method of claim 4, wherein the PDE-inhibiting compound is linked to a targeting molecule.
 7. The method of claim 4, wherein the PDE-inhibiting compound is administered prophylactically to an individual at risk of having a PDE-associated disease or disorder.
 8. The method of claim 4, wherein the PDE-inhibiting compound is administered in combination with an additional drug for treating a PDE-associated disease or disorder.
 9. The method of claim 4, wherein the PDE-associated disease or disorder is a neurodegenerative disorders, penile erectile dysfunction, anxiety, depression, Alzheimer's disease, Parkinson's disease, Huntington's disease, schizophrenia, psychosis, sepsis, asthma, chronic obstructive pulmonary disease, pulmonary hypertension, renal disease, stroke, rhinitis, psoriasis, memory loss, chronic lymphocytic leukemia, prostate cancer, thyroid disease, male hypogonadism, cardiac disease, diabetes, obesity, multiple sclerosis, rheumatoid arthritis, osteoporosis, or cystic fibrosis.
 10. A method for increasing the level of a substrate of a PDE in a cell or tissue, comprising: contacting the cell or tissue with an effective amount of a PDE-inhibiting compound represented by any one of the formulas of groups NI to NIII or an analog, derivative, or variant thereof that inhibits PDE activity, whereby PDE4 activity or PDE7 activity is inhibited and the level of the substrate of the PDE in the cell or tissue is increased.
 11. The method of claim 10, wherein the substrate is cAMP.
 12. The composition of claim 1, wherein the PDE inhibitor comprises: a compound having the formula NI,

wherein: R⁷ and R⁸ can be the same or different and are optionally substituted carbonyl groups; and R⁹ is alkyl.
 13. The composition of claim 12, wherein: R⁷ is C═OO)R^(a), wherein R^(a) is optionally substituted alkyl; R⁸ is (C═O)R^(b), wherein R^(b) is optionally substituted alkyl or optionally substituted arylalkyl; and R⁹ is cyclohexyl.
 14. The composition of claim 12, wherein the compound is:


15. The composition of claim 12, wherein the compound is:


16. The composition of claim 1, wherein the PDE inhibitor comprises: a compound having the formula, NII,

wherein: R¹⁰ is an aryl group, optionally substituted; and R¹¹, R¹², R¹³, R¹⁴, and R¹⁵ can be the same or different and are hydrogen, halide, or alkyl.
 17. The composition of claim 16, wherein: R¹⁰ is phenyl, optionally substituted with N(CH₃)₂ or NO₂; R¹¹, R¹², R¹⁴, and R¹⁵ are hydrogen; and R¹³ is methyl, chloro, fluoro, or bromo.
 18. The composition of claim 16, wherein the compound is:


19. The composition of claim 16, wherein the compound is:


20. The composition of claim 1, wherein the PDE inhibitor comprises: a compound having the formula, NIII

wherein:

is a single bond or a double bond; R¹⁶ and R¹⁷ can be the same or different and are hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heterocycle, or an optionally substituted carbonyl group; or, R¹⁶ and R¹⁷ are joined together to form a ring, optionally substituted; R¹⁸ is alkyl; R¹⁹ is hydrogen, optionally substituted alkyl, or an optionally substituted carbonyl group; or, R¹⁸ and R¹⁹ are joined together to form a ring, optionally substituted; R²⁰ is absent or hydrogen, provided that, when

is a single bond, R²⁰ is hydrogen, and, when

is a double bond, R²⁰ is absent; and R²¹ is ═S, optionally substituted alkyl, optionally substituted heteroalkyl, or optionally substituted arylalkyl.
 21. The composition of claim 20, wherein R¹⁸ is methyl.
 22. The composition of claim 20, wherein R¹⁶ and R¹⁷ are joined together to form an a cycloalkyl or heterocycle ring, optionally substituted.
 23. The composition of claim 20, wherein R¹⁸ and R¹⁹ are joined together to form a cycloalkyl or heterocycle ring, optionally substituted.
 24. The composition of claim 20, wherein R¹⁹ is (C═O)NHR^(c), and R^(c) is an optionally substituted aryl group.
 25. The composition of claim 20, wherein the compound is:


26. The composition of claim 20, wherein the compound is: 